The binding mechanism of Streptomyces trehalase inhibitor SGI and Rhizopus glucoamylase was investigated by the following approaches; inhibitory kinetic study in the steady-state, fluorometric titration, UV difference spectrophotometry, and stoppedflow kinetic study with fluorescence monitoring.
The inhibition by SGI of the hydrolysis of p-nitrophenyl α-D-glucoside was found to be of the mixed type with an inhibitor constant K₁=16.4 μ_M (pH 4.5, 25&C). From fluorometric titration of the enzyme with SGI, the dissociation constant of the enzyme-SGI complex, K_d, was estimated to be 24.5 μ_M (pH 4.5, 10&C). The thermodynamic quantities were obtained from the temperature dependence of K_d, and a large positive entropy change, 74 J•mol^-1 (18 e. u.), and a small negative enthalpy change, -4.2 kJ•mol^-1 (-1.0 kcal•mol^-1), were noted. The pH dependence of K_d suggested that the binding reaction involves one ionizable group of pK_e=5.8 (both at 5&C and at 25&C). The fluorometric titration of the enzyme with gluconic acid-1, 5-lactone in the presence of SGI suggested the binding of SGI to the nonreducing-end terminal subsite (Subsite 1) of the enzyme (Hiromi et al. (1973) Biochim. Biophys. Acta 302, 362). The UV absorption difference spectrum of the enzyme caused by the binding of SGI was similar to that caused by gluconic acid-1, 5-lactone, except that the trough near 300 nm is not as clear in the case of SGI as in the case of gluconic acid-1, 5-lactone.
Fluorescence stopped-flow kinetics of the binding was consistent with a twostep mechanism in which a fast bimolecular association is followed by a slow unimolecular process. The dissociation constant of the fast bimolecular step was estimated to be 4.9mM, and the forward and the backward rate constants of the unimolecular process were determined to be 220 s^-1 and 1.1 s^-1, respectively. It was found that the decrease in the enzyme fluorescence due to the binding of SGI occurs solely in the unimolecular process.

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The product distribution of Brevibacterium ammoniagenes fatty acid synthetase has been investigated using propionyl-CoA instead of acetyl-CoA as the primer. The synthetase produces not only an odd-numbered fatty acid (heptadecanoic acid) but also even-numbered fatty acids (stearic and oleic acids) in the presence of propionyl-CoA. The amounts of heptadecanoic, stearic and oleic acids increased with increasing concentration of propionyl-CoA. However, the formation of heptadecenoic acid (C_17:1) was not observed under any conditions tested. The failure of C_17:1 synthesis suggested that the enzyme component catalyzing the β, γ-dehydration, which is responsible for the synthesis of unsaturated fatty acids, has a high degree of chain length specificity. Under standard assay conditions, stearic acid predominated and heptadecanoic and oleic acids were found in lesser amounts. Mass spectrometric analyses of fatty acids synthesized either from [²H] propionyl-CoA or in ²H₂O revealed that propionyl-CoA is utilized as the priming substrate for the synthesis of heptadecanoic acid and that an acetyl residues, which is formed by the decarboxylation of malonyl-CoA, served as the priming substrate for the syntheses of stearic and oleic acids. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. It is concluded that the decarboxylation of the malonyl moiety bound to the synthetase occurs efficiently only in the course of fatty acid synthesis. A hypothetical scheme is presented to explain the propionyl-CoA-dependent decarboxylation of the malonyl moiety.

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The peptidoglycan-associated lipoprotein (PAL) is present in the cell envelope in a form closely, but not covalently, associated with peptidoglycan in various Gramnegative bacteria. When the cell envelope or the isolated peptidoglycan-PAL complex from Proteus mirabilis, in which PAL maintains the interaction with peptidoglycan, was digested with trypsin, a polypeptide fragment with molecular weight 11, 000 (11 K fragment) was obtained. However, when isolated PAL or the 11 K-fragment which had been dissociated from peptidoglycan was treated with trypsin, they were further digested. The 11 K-fragment maintained essentially the same tight interaction with peptidoglycan as intact PAL. These results indicate that the 11 K-fragment is probably derived from the peptidoglycan-associated region of the PAL molecule. The purified 11 K-fragment contained neither covalently-linked fatty acid nor glycerylcysteine, which are known to be present at the N-terminus of PAL. The N-terminal sequence of the 11 K-fragment was also determined.

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Cholesterol-induced high density lipoprotein (s), termed HDL_c, is one of the abnormal lipoproteins which occur in experimental hypercholesterolemia in several animal species, including rats. Thus far, HDL_c has been exclusively isolated by sequential ultracentrifugation followed by Geon-Pevikon block electrophoresis, which is timeconsuming and requires some specialized knowledge. In this report, a faster and more convenient alternative method for the isolation of HDL_c is described. A combination of a single ultracentrifugation and agarose gel chromatography followed by concanavalin A-Sepharose affinity chromatography was employed. HDLG thus obtained was similar to and probably identical with the HDL_c isolated by Geon-Pevikon electrophoresis, with respect to chemical composition, electrophoretic properties in agarose gel, apoprotein patterns in SDS polyacrylamide gel electrophoresis, and electron micrographic appearance.

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Tokyo Metropolitan Institute of Medical Science, An iso-osmotic Percoll density gradient was applied to determine the subcellular localization of the O₂- generating enzyme, NADPH oxidase, in guinea pig polymorphonuclear leukocytes (PMN). [¹⁴C] Myristate (MA) was employed as a metabolic stimulant in order to clarify whether the myristate binding site on PMN membrane was identical with the O₂- generating site. The distribution pattern of the O₂- generating activity of MA-activated PMN was compared with that of unactivated PMN in parallel experiments to find fractions showing an enhanced O₂- generating activity (i.e., above the background values due to O₂- generation by other electrontransport systems). We observed very high O₂ generating activity concentrated in a single peak with MA-activated PMN but little activity was seen with unactivated PMN in the Percoll density gradient. The O₂- generating activity of MA-activated PMN was consistently associated with 5'-nucleotidase activity as a membrane marker enzyme, but was not associated with lysosomal marker enzymes such as myeloperoxidase and lysozyme. O₂- generating and 5'-nucleotidase activities in the peak fraction of MA-activated PMN were increased to about six and four times those of whole cells in terms of specific activity, respectively. These results indicate that the O₂- generating enzyme is located on PMN plasma membrane. Furthermore, [¹⁴C] myristate-binding activity was mainly found in the peak fraction containing O₂- generating enzyme. This suggests that [¹⁴C] myristate binds to plasma membrane, and the O₂- generating enzyme may thus be activated.

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Highly purified uricase [urate: oxygen oxidoreductase, EC 1. 7. 3. 3] was obtained from Candida utilis by affinity chromatography with xanthine-agarose conjugate followed by chromatography with Sephadex G-200 in the presence of dithiothreitol. The uricase molecule had a molecular weight of 120, 000 and was composed of four identical subunits with a molecular weight of 30, 000. The amino acid composition was determined and the N-terminal amino acid was identified as methionine. Other physicochemical properties obtained were as follows; isoelectric point (5. 6), α-helix content (12%; b₀=-76±15&), specific activity (25.8 units/mg protein) and K_m value (1.0×10^-5M) for uric acid at pH 8.5 and 25&C. The uricase preparation did not contain detectable amounts of the following metals; Fe, Cu, Mg, Mn, Co, Ni, Zn, and Cd. There were three cysteine residues in the subunit molecule and these were classified into two types by a chemical modification study; one is accessible to a reagent and the remaining two become accessible only after denaturation. The former may be located in the vicinity of the active center of the molecule, although it does not participate directly in the catalysis.

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Muscle, heart (brain) and liver type isoenzymes of glycogen phosphorylase α [EC 2. 4. 1. 1] were purified to homogeneity from the pig skeletal muscle, heart and liver. Dephosphorylations of these isoenzymes by phosphoprotein phosphatases [EC 3. 1. 3. 16] with a molecular weight of 224, 000 purified from these pig tissues were studied. Apparent K_m values of phosphoprotein phosphatases from the skeletal muscle, heart, and liver for the homologous tissue type isoenzymes were 8.8, 9.0, and 8.8 μM, respectively. Apparent V_max values of dephosphorylation were 2.5, 6.5, and 1.2 nmol/min per unit, respectively (the unit was defined with rabbit skeletal muscle phosphorylase α as a substrate). Inhibition of dephosphorylation of the heart type isoenzyme by glucose 6-phosphate was partially competitive with a K₁ value of 0.05mM. Though dephosphorylation of the liver type isoenzyme was inhibited competitively by glucose 6-phosphate with a K₁ value of 3.5mmM, dephosphorylation of the muscle type isoenzyme was not affected by 0.1-1mM glucose 6-phosphate. AMP was a strong competitive inhibitor of the dephosphorylation reactions with respect to substrate isoenzymes and also served as an allosteric effector of the heart type isoenzyme. AMP concentrations required for 50% inhibition were 2 μM with the muscle type isoenzyme, 1 μM with the heart type isoenzyme, and 38 μM with the liver type isoenzyme. Differences in V_max values and in degrees of inhibition by the metabolites of dephosphorylation of the isoenzymes were observed with phosphoprotein phosphatases from either homologous or heterologous tissues indicating that these differences were mainly attributed to phosphorylase α isoenzymes. A method for the purification of phosphorylase b from pig liver containing a low level of glycogen was developed.

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The effects of native and modified bovine serum albumins on the esterification of cholesterol in sonicated dispersions of lecithin-cholesterol mixtures by lecithincholesterol acyltransferase [EC 2. 3. 1. 43] (LCAT) in human plasma were studied in the presence or absence of Cd²⁺ ion.
As the modified albumins, 52%- and 84%-acetylated, 48%- and 82%-succinylated, and 24%- and 63%-glycine methylesterified albumins were prepared chemically. These modified albumins still contained native structure of serum albumin, as revealed by intrinsic viscosity, ultraviolet absorption and circular dichroism measurements.
The acyltransferase activity was stimulated by the addition of native albumin in vitro. This stimulatory action of native albumin was reduced by acetylation or succinylation of its amino groups. However, no inhibitory action of acetylated and succinylated albumins was observed. On the other hand, the stimulatory action of native albumin completely disappeared upon methylation¹ and glycine methylesterification of its carboxyl groups. In contrast, the esterified albumins showed a potent inhibitory action on the acyltransferase. The inhibitory action of these esterified albumins with apparently increased positive charges was greater than that of poly-L-lysine, which has many positive charges of the molecule.
On the other hand, the acyltransferase activity was decreased by the addition of 1×10^-3M Cd²⁺ ion. The decreased acyltransferase activity in the presence of Cd²⁺ ion recovered progressively on the addition of increasing amounts of native, acetylated, and succinylated albumins. In particular, the extent of recovery of the decreased acyltransferase activity produced by acetylated and succinylated albumins in the presence of Cd²⁺ ion was greater than that with native albumin. However, no recovery effect of the esterified albumins on the decreased acyltransferase activity was observed in the presence of Cd²⁺ ion. On the contrary, additive inhibitory actions of Cd²⁺ ion and esterified albumin on the acyltransferase activity were observed.

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The immunological properties of thiol proteinases from rat liver, cathepsins B, H, and L, were examined. Rabbit antisera were prepared against cathepsins B and H from rat liver. Anti-cathepsin B and H sera quantitatively precipitated only the corresponding enzyme protein. On immunodiffusion analysis, anti-cathepsin B serum reacted with cathepsin B, but not with cathepsin H or cathepsin L, and similarly anti-cathepsin H reacted only with cathepsin H, indicating that the three thiol proteinases are distinct. Immunoelectrophoresis using a mixture of anti-cathepsin B and anti-cathepsin H showed that the precipitin line with cathepsin B moved faster to the anode than that with cathepsin H. Immunological diffusion with antisera indicated that rat liver cathepsins B and H were immunologically identical with cathepsins B and H from rat kidney, lung, spleen, brain, and heart, but different from cathepsin B or H from human or rabbit liver.

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Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37&C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.

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A new method is reported for preparing a thin planar membrane of 1, 2-distearoyl-sn-glycero-3-phosphocholine and egg yolk lecithin-cholesterol (molar ratio of 1:1) between a pair of cellulose sheets. This technique, developed from the method of the multilayer planar membrane preparation (Setaka, M., et at. (1979) J. Biochem. 86, 355-362; 1619-1622; (1980) J. Biochem. 88, 1819-1829), consisted of three experimental processes. First, a phospholipid monolayer was prepared at an air-water interface, then taken up on a stretched cellulose sheet. A thin lipid membrane, supported from both sides by cellulose sheets, was constructed by combining two of these lipid monolayer-cellulose sheets.
The permeability coefficient of the thin lipid membrane was estimated by removing the effect of two outer cellulose sheets, and this permeability was found to be larger than those of other model membranes of a lipid bilayer, indicating that the present lipid membrane is not a perfect single lipid bilayer. However, certain experimental evidence suggests that the bulk of the phospholipids formed a bilayer between the two cellulose sheets.
Since this lipid membrane is particularily stable, larger membranes can be prepared by the present method than other planar bilayer membranes of lipid, which are usually constructed inside a pin hole in a thin teflon sheet.

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Membrane-bound neutral proteinase was found in the microsomal fraction of rat skeletal muscle as assayed with heat-denatured casein as a substrate. The enzyme was solubilized from 1_M KCl-washed microsomal fraction by 1% sodium cholate containing 0.15_M NaCl, and partially purified by chromatography on a column of Sepharose CL-6 B in the presence of 0.5% sodium cholate and 0.1_M NaCl. The enzyme was eluted from the Sepharose column as a single but rather broad peak at a position corresponding to a molecular weight of about 190, 000. The pH optimum for hydrolysis of heat-denatured casein was about 8.0. It was inhibited to significant extents by various reagents including diisopropyl phosphorofluoridate, phenylmethanesulfonyl fluoride, N^α-tosyl-L-phenylalanine chloromethyl ketone, N^α-tosyl-L-lysine chloromethyl ketone, p-chloromercuriphenyl sulfonate, chymostatin, EDTA, EGTA, and o-phenanthroline. This inhibition profile suggests that the present muscle proteinase is a mixture of proteinases, such as a serine proteinase and a metallo-proteinase similar to those occurring in the microsomal membranes of liver and kidney (or small intestine), respectively. Among urea-denatured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by protamine, hemoglobin, and casein.

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Sodium- and potassium-activated adenosinetriphosphatase (Na⁺, K⁺-ATPase) purified from dog kidney outer medulla was examined by polyacrylamide gel electrophoresis and by photoaffinity labeling with N-(ouabain)-N'-(2-nitro-4-azidophenyl)-ethylenediamine (NAP-ouabain).
The large subunit band (α-band) split into two bands on the gel after the enzyme was heat-treated in the presence of 1% sodium dodecylsulfate (SDS). Of the two bands (αI and αII), αI had the same electrophoretic mobility as the original band, while αII moved slightly faster. Total conversion into αII was not observed, about half of the original remaining as αI. Below 60&C, heat treatment did not produce αII. Phenylmethylsulfonyl fluoride did not prevent the appearance of αII.
Both αI and αII were labeled with [³H] NAP-ouabain. Nonspecific incorporation of [³H] NAP-ouabain also occurred irrespective of illumination, but it was removed either by diffusion during staining and destaining of the gel or by treatment of the enzyme with trichloroacetic acid.
It is tentatively concluded that the splitting of the band reflects some intrinsic differences in situ of the α-subunit of dog kidney membrane Na⁺, K⁺-ATPase.

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The site of biosynthesis of sulfite oxidase in rat liver was determined by immunoprecipitation of nascent peptides released from ribosomes and in vitro translation products of isolated polysomes. The specificity of the immunoprecipitation of sulfite oxidase nascent peptides was confirmed by examination of the size distribution of immunoprecipitated peptides and by the competition with purified enzyme in the immunoprecipitation. Both nascent peptides analysis and the in vitro translation experiment indicated that this enzyme was mainly synthesized by free ribosomes in the cytoplasm, although bound ribosomes also contributed to the synthesis of this enzyme to some extent. When the discharge of sulfite oxidase peptides from bound ribosomes was examined with rough microsomes, however, about two thirds of the enzyme peptides synthesized by bound ribosomes were discharged to the outside of microsomal vesicles.

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The time courses of in vivo incorporation of [³H] leucine into mitochondrial, microsomal, and cytosolic sulfite oxidases were examined and it was found that the cytosol contained a precursor pool of newly synthesized sulfite oxidase which was rapidly transported into mitochondria. At 10min after the injection of the radioactive leucine, most of the radioactivity of cytosolic sulfite oxidase was associated with the precursor whose molecular size was about 3, 000 daltons larger than the authentic sulfite oxidase subunit. We concluded that sulfite oxidase was synthesized as a larger precursor in the cytosol, and transported into mitochondria to be converted to the mature enzyme. The turnover rate of sulfite oxidase in rat liver was also determined by measuring the decay of enzyme radioactivity after the injection of [¹⁴C] leucine. Mitochondrial, microsomal, and cytosolic sulfite oxidase had an almost identical half-life of 3-4 days.

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1. In order to elucidate the structure-function relation of a glucoamylase [EC 3. 2. 1. 3, α-D-(1→4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M₁), the reaction of Gluc M₁ with water-soluble carbodiimides was studied.
2. Gluc M₁ was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide (CMC) at pH 4.5.
3. Inactivation of Gluc M₁ with [¹⁴C] CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M₁.
4. The CD spectrum of CMC-modified Gluc M₁ (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained.
5. In the presence of maltose, when Gluc M₁ was incubated with [¹⁴C] CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M₁ modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M₁ was inactivated completely with the incorporation of ca. 4 CMC moieties.
6. The logarithm of the half-life of the inactivation of Gluc M₁ by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M₁.
7. The protection by maltose of Gluc M₁ from inactivation and the increase in K₁ values for maltose of CMC-modified Gluc M₁'s suggested that a crucial carboxyl group (s) was located near or on subsites 2 and 3.

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Physicochemical experiments were performed in order to get further evidence of the formation of complexes between iron-sulfur proteins and flavoproteins from spinach chloroplast and beef adrenal cortex electron-transfer systems.
With both spinach and adrenal iron-sulfur protein-flavoprotein systems, characteristic difference circular dichroism (CD) spectra between the sum of CD spectra of the respective proteins and the CD spectrum of a mixture with a molar ratio of one to one were obtained in the visible and far-ultraviolet regions. By differential scanning calorimetry (DSC) measurements, ferredoxin and ferredoxin-NADP reductase were found to be stabilized against heat treatment upon mixing: (1) the apparent denaturation temperatures of ferredoxin and the reductase increased by about 18 and 5&C, respectively, and (2) the activation energy of the denaturation reaction increased by about 50%.
These results lead to the conclusion that the iron-sulfur protein and flavoprotein form a tightly bound complex, and suggest that the complex formation alters both the environments of the iron-sulfur and flavin chromophores and the contents of the ordered structures, resulting in stabilization of the proteins.

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Human lactoferrin contains 2 asparagine-linked sugar chains in 1 molecule. These sugar chains were released as oligosaccharides by hydrazinolysis from two lactoferrin samples of different races. The two oligosaccharide fractions gave exactly the same fractionation pattern upon paper electrophoresis and Bio-Gel P-4 column chromatography after sialidase digestion. A structural study of the oligosaccharides obtained from the two samples by sequential exoglycosidase digestion in combination with methylation analysis also gave the same results indicating that there is no racial difference both in quality and quantity of the sugar chain moiety of lactoferrin. In addition to the two acidic sugar chains, NeuAcα2→6 Galβ1→4 GlcNAcβ1→2 Manα1→6 (NeuAcα2→6 Galβ1→4 GIcNAcβ1→2 Manα1→3) Man β1→4 GlcNAcβ1→4 (Fucα1→6) GlcNAc and Galβ1→4 (Fucα1→3) GlcNAcβ1→2 Manα1→6 (NeuAcα2→6 Galβ1→4 GlcNAcβ1→2 Manα1→3) Manβ1→4 GlcNAcβ1β4 (Fucα1→6) GlcNAc, and two novel acidic sugar chains, Galβ1→4 (Fucα1→3) GlcNAcβ1→2 Manα1→6 (NeuAcα2→6 Galβ1→4 GlcNAcβ1→3 Galβ1→4 GlcNAcβ1→2 Manα1→3) Manβ1→4 GlcNAcβ1→4 (Fucα1→6) GlcNAc and Galβ1→4 (Fucα1→3) GlcNAcβ1→3 Galβ1→4 GlcNAcβ1→2 Manα1→6 (NeuAcβ2→6 Galβ1→4 GlcNAcβ1→2 Manα1→3) Manβ1→4 GlcNAcβ1→4 (Fucα1→6) GlcNAc, were found to occur in human lactoferrin.

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The mechanism of ATP hydrolysis by chicken gizzard myosin was investigated at 0.2M KCl where dimerization of myosin occurs as well as at 0.3M KCl where dimerization of myosin does not occur.
1. The ATP-induced difference spectrum of UV absorption of gizzard myosin obtained in 0.2M KCl was identical, not only in size but also in shape, with that obtained in 0.3M KCl. Therefore, we propose that the ATP-induced conformation of myosin is the same in both its dimeric form and its monomeric form.
2. According to a kinetic study, the rate of the ATP-induced enhancement in myosin fluorescence obtained in 0.2M KCl was approximately equal to that obtained in 0.3M KCl. On the other hand, the rate of the subsequent decay of the ATP-induced fluorescence enhancement was much lower in 0.2M KCl than in 0.3M KCI.
3. The rate of the initial burst of ATP hydrolysis obtained in 0.2M KCl was approximately equal to that obtained in 0.3M KCl. On the other hand, the rate of ATP hydrolysis in the steady state was much lower in 0.2M KCl than in 0.3M KCl. Based on the results described above, we propose (a) that (M^**ADP_P)₂ can be degraded only via monomers of M^**ADP_P and (b) that the concentration of monomers of M^**ADP_P in the steady state of ATP hydrolysis is decreased as a consequence of the dimerization of M^**ADP_P in 0.2M to KCLl. (^** indicates the conformational change of myosin with the spectrum change of UV absorption.)
We also found that upon addition of AMPPNP, a dimeric form of gizzard myosin was produced in 0.2M KCl. Therefore, ATP hydrolysis appears not to be required for the formation of myosin dimers.

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1. Guanine deaminase [EC 3. 5. 4. 3] was purified to a homogeneous state from rat brain by a procedure involving ammonium sulfate fractionation, DE-52 column chromatography, hydroxylapatite column chromatography, gel filtration on ACA-34 and isoelectric focusing. Homogeneity was shown by polyacrylamide gel electrophoresis in the presence and absence of SDS.
2. The molecular weight of the enzyme was determined by gel filtration (105, 000), and that of its subunit by SDS-polyacrylamide gel electrophoresis (52, 000). From these findings, we concluded that the native enzyme consisted of two identical subunits.
3. The K_m values for guanine and 8-azaguanine were calculated to be 0.17mM and 0.67mM, respectively. This enzyme was markedly inhibited by 5-amino 4-imidazolecarboxamide (AICA), a precursor of purine nucleotide synthesis, with a K₁ value of 82 μM.
4. Guanine deaminase was purified from rat liver by the procedure used for purification of the brain enzyme and anti-liver enzyme serum was raised in rabbits. This antiserum cross-reacted with the brain enzyme without spur formation in the Ouchterlony double diffusion test.
5. The γ-globulin fraction of the anti-guanine deaminase serum and AICA inhibited more than 50% of the ammoniagenesis in the brain system in which the purine nucleotide cycle operates. It was also shown that guanine nucleotides were degraded via guanosine and guanine liberating ammonia in the same brain system when substrates for the purine nucleotide cycle were omitted.
On the basis of these findings it is suggested that guanine deaminase contributes to ammoniagenesis in the brain.

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Phosphoprotein phosphatases [EC 31. 3. 16] acting on P-H 2 B histone, P-H 1 histone, phosphorylase α [EC 2. 4. 1. 1] and glycogen synthase b [EC 2. 4. 1. 11] were surveyed in cytosols from various rat tissues and erythrocytes by gel-filtration on Sephadex G-200. A main phosphoprotein phosphatase (Mr=200, 000) in tissue cytosols showed preference for phosphorylated histones. In contrast to tissue cytosols, erythrocyte cytosol contained a major phosphoprotein phosphatase (Mr=100, 000) which showed strong preference for phosphorylase α and synthase b. Other phosphoprotein phosphatases which preferred phosphorylase α were detected in cytosols from liver, kidney, spleen, and adipose tissue. The molecular weights of the phosphatases were estimated to be 140, 000 for liver enzyme and about 90, 000 for kidney, spleen, and adipose tissue enzymes. A phosphoprotein phosphatase (Mr=54, 000) which preferred synthase b and P-H 2 B histone was also found in cytosols from various tissues and erythrocytes. This phosphatase was most abundant in liver cytosol and showed strong dependency on Mg²⁺ and Mn²⁺. This enzyme was different from other phosphoprotein phosphatases in specificities for different phosphorylated sites on glycogen synthase b. Although treatment with 80% ethanol did not affect the size of liver 54, 000 molecular weight enzyme, the same treatment lowered the molecular weights of the major erythrocyte enzyme (Mr=100, 000), the main skeletal muscle, liver, and kidney enzymes (Mr=200, 000), liver 140, 000 molecular weight enzyme and kidney 93, 000 molecular weight enzyme to about 30, 000. As a result of this conversion, P-H 1 histone phosphatase activities and Mg²⁺ requirements for P-H 2 B histone phosphatase activities of these enzymes were greatly suppressed. Phosphorylase phosphatase activities of the 200, 000 and 140, 000 molecular weight enzymes were significantly enhanced, but the activity of the 100, 000 molecular weight enzyme was essentially unchanged and that of the 93, 000 molecular weight enzyme was slightly suppressed. Synthase phosphatase activities of the 200, 000 molecular weight enzymes increased 2-3-fold, but the activities of the other enzymes were either unaffected or insignificantly suppressed. The 30, 000 molecular weight enzymes derived from these enzymes showed same physical and catalytic properties, suggesting that the multiple molecular forms of phosphoprotein phosphatases (except for the 54, 000 molecular weight enzyme) contained a common 30, 000 molecul_??_r weight catalytic subunit(s).

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Tunicamycin (TM), an antibiotic that blocks glycosylation of glycoproteins by inhibiting the formation of dolichyl N-acetylglucosaminyl pyrophosphate was used to study the expression of active repressible acid phosphatase (r-APase) [EC 3. 1. 3. 2] by yeast protoplasts. Secretion of active r-APase was completely inhibited by TM, however, neither accumulation of r-APase activity inside protoplasts nor inhibition of protein synthesis were observed on TM-treatment. The results led us to postulate that an enzymatically inactive form of nonglycosylated r-APase is accumulated in the membrane fraction on TM-treatment. Protoplasts of various r-APase mutants were radiolabeled with [S³⁵] methionine in the presence or absence of TM, then membrane fractions were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and an autoradiogram was prepared. In r-APase-producing protoplasts of the wild type strain, the membrane (100, 000×g sedimentable) fraction of TM-treated protoplasts contained, in addition to usual membrane proteins, three proteins with molecular weights of 59 K, 57 K, and 55 K daltons. The three proteins are also formed in TM-treated protoplasts of the pho80 mutant constitutively forming r-APase in the presence of P₁. However, TM did not induce formation of these three proteins in the protoplasts of r-APase negative mutants (pho 2, pho 4, and pho 81), or in the protoplasts of temperature sensitive mutants (pho 2^ts and pho 4^ts) at a nonpermissive temperature (35&C), or in TM-treated protoplasts of the wild type strain repressed by P₁. The three proteins were located in the membrane fraction and not in the cytosol or outside of the protoplasts. The purified r-APase preparation deglycosylated by hydrogen fluoride (HF) treatment at 0&C for 1 h or by endo-β-N-acetylglucosaminidase H (Endo H) treatment, yielded three proteins with molecular weights of 62 K, 60 K, 58 K or 60 K, 58 K, 56 K daltons, respectively. We considered the purified r-APase preparation to be composed of three different molecules, which yielded three deglycosylated proteins. The difference of about 2, 000 daltons in apparent molecular weight between the deglycosylated proteins produced by HF and Endo H treatments is probably due to N-acetylglucosamine residues attached to asparagine residues in the r-APase proteins.
Our results indicate that the 59 K, 57 K, and 55 K proteins produced by TM-treatment are nonglycosylated forms of r-APase and that addition of core-oligosaccharide is required for formation of active r-APase and its secretion by yeast protoplasts.

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Alkaline phosphatase was purified from plasma membrane of rat ascites hepatoma AH-130 cells by chromatographies on DEAE-cellulose and Affi-Gel Blue and preparative polyacrylamide gel electrophoresis. The yield of the purified enzyme, finally as the denatured subunits, was about three times better than that obtained previously [J. Biochem. (1978) 83, 1471-1483]. The purified enzyme, a glycoprotein, was analyzed for amino acid and carbohydrate compositions. The composition of the carbohydrate moiety, which amounted to about 18% by weight, indicated that both N- and O-glycosidic sugar chains were contained in the protein.
The purified alkaline phosphatase (as the denatured subunits) was used to produce morospecific antibody, which was found to have the same immunological reaction with the native antigen as the antibody raised against the partially purified native enzyme. Immunological analysis by Ouchterlony double gel diffusion and quantitative immunoprecipitation demonstrated that the hepatoma enzyme was identical with that of the liver.
In standard polyacrylamide gel electrophoresis at pH 8.9, the hepatoma alkaline phosphatase migrated a little more slowly than the liver enzyme. Both enzymes, however, showed identical mobility after complete removal of sialic acid from them with neuraminidase. These results suggest that the only difference between the two enzymes was in the carbohydrate moiety, especially in the sialic acid content.

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Glycerol dehydrogenase [EC 1. 1. 1. 6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20mM acetate buffer (pH 6.0) on either octyl-Sepharose or 10-carboxydecyl-Sepharose, among ten different kinds of n-alkyl-Sepharose derivatives tested, some of which have an amino or a carboxyl group as a terminal residue.
The enzyme adsorbed on 10-carboxydecyl-Sepharose is 95% eluted with 0.26M NaCl. n-Propanol (10% and 15%, but not 5%), and various nucleotides such as NAD^-, NADH, NADP⁺, NADPH, AMP, ADP, and ATP (1mM) are also effective for elution. The elution with nucleotides is facilitated by 5%, n-propanol. On the other hand, the enzyme adsorbed on octyl-Sepharose is not eluted under the conditions described above.
These results suggest that the adsorption-elution of the enzyme on 10-carboxydecyl-Sepharose is due to a combination of hydrophobic interaction and electrostatic repulsion between a specific locus of the enzyme surface and the 10-carboxydecyl residue.
Experimental conditions are described under which the enzyme can be purified 266-fold with a yield of 79% by a single chromatography of the cell extract on a 10-carboxydecyl-Sepharose column.

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The effects of the administration of glucagon and a high-protein diet, the conditions for the induction of rat liver serine: pyruvate aminotransferase, on the subcellular distribution of this enzyme were studied.
There were two classes of serine: pyruvate aminotransferase activity which were distinguishable by the reactivity with a specific antibody prepared against purified serine: pyruvate aminotransferase. In the liver of fasted rats, the aminotransferase activity was located in peroxisomes, mitochondria and the cytosolic fraction in a ratio of 23:49:28 as judged by primary subcellular fractionation and subsequent ultracentrifugation on a sucrose density gradient. In mitochondria and the cytosolic fraction, however, the immunoreactive activity accounted for only approximately 30% and 50% of the total activity in the respective fractions, while over 90% of the peroxisomal activity reacted with the antibody. The percentage distribution of serine: pyruvate aminotransferase in peroxisomes, mitochondria and the cytosolic fraction was thus calculated to be 40, 30, and 30%, respectively.
Only the mitochondrial serine: pyruvate aminotransferase activity was remarkably increased after the administration of glucagon, and when only the immunoprecipitable activity was concerned, the increase was as much as approximately 100-fold after 3 daily injections of the hormone. The concomitant rise in the immunoreactive activity in peroxisomes and the cytosolic fraction was several fold, suggesting the existence of some mechanisms which permit the selective accumulation in mitochondria of serine: pyruvate aminotransferase synthesized under the acute effect of glucagon. In the liver of rats fed on an 88% casein diet for 8 days, the hepatic activity of serine: pyruvate aminotransferase increased approximately 3-fold, but the preferential increase in the activity of any particular subcellular fraction was not as clearly seen as in the case of the liver of glucagon-treated rats.
Since rat liver serine: pyruvate aminotransferase has a relatively broad substrate specificity, the enzyme induced under the conditions of glucagon abundance appeared to participate in the catabolism of various neutral amino acids, such as phenylalanine, asparagine and glutamine, in addition to that of serine.

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The paper electrophoretic pattern of the oligosaccharides released from the plasma membranes of K-562 cells by hydrazinolysis was quite different from that of human erythrocyte membranes. Bio-Gel P-4 column chromatography in combination with sequential exoglycosidase digestion of the neutral oligosaccharide fractions revealed that all those from K-562 cells are of the high mannose type, while those from erythrocytes are of large complex type structures.
Studies of the acidic oligosaccharides indicated that none of those obtained from K-562 cells contained the β-N-acetylglucosamine residue linked at the C-4 position of the β-mannosyl residue of the trimannosyl core, which occurs in most of the asparagine-linked sugar chains of human erythrocytes. This indicates that the glucosaminyltransferase that forms the GlcNAcβ→4 Manfβ1→4 group has not been expressed in K-562 cells.

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The accuracy of radioimmunoassay (RIA) has been much improved for sea-squirt antigen Gi-x, which is considered to be of higher molecular weight than antigen Ei-M, by using radiolabeled Gi-x as a tracer. Gel chromatography monitored by the improved RIA revealed a wide distribution of molecular weight of Gi-x type antigens, in contrast to Ei-M. However, since a considerable portion of the antigenic activity gave a single peak in gel chromatography with Sepharose 6 B, the substance in the peak fractions was isolated as a fairly homogeneous preparation and referred to as Gi-rep. Gi-rep showed distinct characteristics of Gi-x type antigens and was clearly discriminated from Ei-M by radioimmunometry in vitro. The in vitro observation also suggested that Gi-rep and Ei-M carried a common antigenic determinant (type α), but that Ei-M also carried a specific determinant (type β). The weight-average molecular weights were 1.1×10⁵ for Gi-rep and 2.3×10⁴ for Ei-M. Both preparations consisted of acidic glycoproteins with considerable amounts of sulfate and phosphate.

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Purothionin from wheat flour was chemically modified by acetic or succinic anhydride under specific conditions. The complete modification of all amino groups of purothionin caused a large change in the net charge of the molecule, leading to the loss of the toxicity to mice and yeast. The sole tyrosyl residue in purothionin was nitrated by tetranitromethane at neutral pH or iodinated by the lactoperoxidase method. The nitro- and diiodo-derivatives of purothionin showed considerably reduced toxicity. Based on these modification studies we conclude that the positive charges of lysyl residues have an important role in the interaction with the negatively charged cell surface, and that the emergence of the toxicity of purothionin depends on a certain state of the tyrosyl residue.

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Sepharose 4 B conjugated with phosphorylated myosin light chains was used in affinity chromatography of a partially purified preparation of gizzard myosin lightchain phosphatase (MLCP) (Onishi et al. (1979) J. Biochem. 86, 1283-1290). The MLCP preparation thus purified contained, according to SDS gel electrophoresis, three components of 67, 000 (67 K), 54, 000 (54 K), 34, 000 (34 K) daltons. In an accompanying report, Uchiwa et al. (J. Biochem. 91, 273-282 (1982)) described the purification of gizzard myosin light-chain kinase, which consisted of two subunits; 130 K and 17 K daltons.
Using the purified preparations of MLCP and MLCK, it was demonstrated a) that reversible changes in the ATPase and superprecipitation activities occur as myosin light chains are enzymatically phosphorylated and dephosphorylated, and b) that addition of a very low concentration of Ca²⁺ and its removal cause reversible changes in the turbidity of actomyosin suspensions as well as in the state of phosphorylation of myosin light chains only when MLCK and MLCP are both present. These results provide strong support for the proposal (see Ikebe et al. (1977) J. Biochem. 80, 299-302) that MLCK and MLCP play a key role in the Ca²⁺ regulation in gizzard.

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1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130, 000 (130 K)-dalton subunit and 17, 000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca²⁺ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region.
2. The Ca²⁺ and Sr²⁺ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of “reconstituted” myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of “reconstituted” myosin B were practically identical with “natural” myosin B in the Ca²⁺ and Sr²⁺ requirements for the three activities measured above.
3. Both the extent and the activity of superprecipitation, and both the initial and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring ³²P incorporation from [γ-³²P] ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.

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The bacterial distribution of D-glucosaminate dehydratase [EC 4. 2. 1. 26] was investigated and Agrobacterium radiobacter (IAM 1526) was found to have the highest enzyme activity. The enzyme was formed inducibly in a glycerol-urea medium by D-glucosaminate, D-galactosamine, and D-glucosamine, but not by D-mannosamine. The enzyme purified from the cells grown in the glucosamine-glycerol-urea medium was shown to be homogeneous by ultracentrifugation. The molecular weight was determined to be about 66, 000 by the sedimentation equilibrium method, and 72, 800 by the gel permeation chromatography low-angle light scattering method. The pH optimum is 8.3-9.0. The enzyme catalyzed the dehydration of D-glucosaminate (relative activity: 100, K_m: 2.8mM), D-galactosaminate (31.5, 5.0mM), D-mannosaminate (17.5, 29mM), D-threonine (5.1, 4.8mM), D-serine (3.2, 0.026mM), and L-serine (1.1, ND), but not L-threonine. The reverse reaction does not occur. The enzyme is inhibited by typical inhibitors of pyridoxal 5'-phosphate enzymes, such as L-penicillamine, and also by carbonyl reagents, thiol reagents, divalent metals, and several D-amino acids and D-amino sugars.

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The present investigation was undertaken to see to what extent the α-amino group of the amino acid, the side chain of the amino acid of aminoacyl-tRNA, and the tRNA structure are involved in determining the affinity of aminoacyl-tRNA for bacterial elongation factor Tu-GTP complex. Various aminoacyl-tRNAs, misaminoacylated tRNAs, and formylated aminoacyl-tRNAs were prepared, and the dissociation constants of the ternary complexes of aminoacyl-tRNA with EF-Tu: GTP were determined by the RNase-resistance assay. The results indicated that the free α-amino group of the amino acids in aminoacyl-tRNA is strongly required for binding with EF-Tu: GTP. In this connection, the biological significance of formylation for Met-tRNA^Met_f species is discussed.

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One of the TLMA (rat T-lymphocyte-macrophage-associated antigens) was characterized as asialo GM 1, Galβ, 1-3) GalNAc (β, 1-4) Gal(β, 1-4) Glc (β, 1-1)-Cer by two assay methods using monospecific anti-asialo GM 1 antibody, immunocytotoxicity testing and immunostaining.
Nylon column-enriched T lymphocytes (non-adherent cells) were highly cytolyzed by anti-asialo GM 1 antiserum and complement as compared with adherent cells (non T cells). Forty % of peritoneal exudate macrophages and 20% of granulocytes were also cytolyzed by anti-asialo GM 1 antiserum. On the other hand, 5% of thymic cells and 60-80% of lymph node T cells were immunostained by antiasialo GM 1 antibody. These asialo GM 1-positive cells in thymus were found to be localized in the cortico-medulla junction. About 20% of macrophages and 2)% of granulocytes were also immunostained by anti-asialo GM 1 F (ab') 2 fragment. These results strongly support the previous conclusion that TLMA can be identified as asialo GM 1. Asialo GM 1 was universally expressed on a certain population of macrophages of various tissues, but not on all of the population. Alveolar macrophages were also asialo GM 1-positive.

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The RNA-dependent DNA polymerase (the reverse transcriptase) was solubilized from three related strains of avian sarcoma virus (ASV B 77, ASV tsLA 334, and ASV QV 2) as well as avian myeloblastosis virus (AMV) and a chicken endogenous virus (RAV-0), by a combination of non-ionic detergent treatment and CsCl stepgradient centrifugation, and was subsequently separated into individual enzyme forms by poly (C)-agarose column chromatography. The newly developed two-step method allowed us to purify the three molecular forms (α-, αβ, and β-form) of highly active enzyme rapidly and quantitatively from all the five virus strains examined. The molar ratio of the three enzyme forms differed among the virus strains: For the three sarcoma viruses, the major species was the αβ-form enzyme, the putative holoenzyme, and the α- and β-form enzymes were less than a few percent and 15-25%, respectively, while the α-form enzyme content was higher for the two leukosis viruses than for the three sarcoma viruses. Both the total DNA polymerase activity and the content of the two enzyme subunits in purified virions of the three sarcoma virus was in the following order: ASV tsLA 334>ASV B 77>ASV QV 2, which paralleled the virus yield at a permissive temperature in roller bottle cultures of chick embryo fibroblasts. No alteration was found in the thermolability of DNA polymerases between tsLA 334, which carries ts mutations affecting both virus growth and cell-transformation, and other viruses.

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The three enzyme forms (α-, αβ-, and β-form) of the RNA-dependent DNA polymerase (the reverse transcriptase) from three strains of avian sarcoma virus (ASV B 77, ASV tsLA 334, and ASV QV 2) and one exogenous (avian myeloblastosis virus (AMV)) and one endogenous avian leukosis virus (Rous-associated virus type-0 (RAV-0)) were compared with each other in subunit structure and catalytic properties.
Despite the gross similarity in the subunit molecular weight (Mr(α)=65, 000 and Mr(β)=92, 000), minor differences were found in the molecular size of the subunit as determined by SDS-gel electrophoresis, the order being ASV tsLA 334≤ASV QV 2<ASV B 77≤RAV-0=AMV. The structural differences were supported by analysis of peptide fragments after treatment with S. aureus V 8 protease. Although the general catalytic properties of the purified enzymes from the five virus strains were similar, the selectivity of template-primer differed in the RAV-0 enzymes. The template-primer selectivity of the reverse transcriptases from all five virus strains tested was also found to be greatly influenced by the reaction temperature for DNA synthesis, resulting in a temperature-dependent increase of poly (dG) synthesis over [(A)_n]•[(dT)_12-18]-dependent poly (dT) synthesis. The RAV-0 enzymes required a lower temperature for DNA synthesis, particularly for [(C)_n]•[(dG)_12-18]-dependent poly (dG) synthesis.

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The influence of oligodeoxynucleotide chain length on the priming activity of the reverse transcription was investigated in poly (A)-dependent poly (dT), poly (C)-dependent poly (dG) and influenza virus RNA-dependent cDNA synthesis by the αβ-form enzyme of the RNA-dependent DNA polymerase from avian sarcomaleukosis viruses. The minimum chain length required for the priming activity to be exposed varied depending on the nucleotide sequence of the primer and thus of the primer-binding sequence of the template RNA, i.e., a sudden and maximal increase of the priming activity was observed at a primer chain length of between 2 and 4 for poly (dG) and between 6 and 8 for poly (dT) synthesis. The observed difference in the minimum chain length seems to parallel the difference in physical stability between poly (C)-oligo (dG) and poly (A)-oligo (dT) hybrids, although the primer chain lengths are shorter than those required for the respective hybrids paired at the reaction temperature.
In influenza virus RNA-dependent cDNA synthesis, the priming activity of oligonucleotides complementary to the 3' terminal sequence of vRNA segments was enhanced at a chain length of between 10 and 12, maintaining a plateau thereafter. This unexpected requirement for long primers in influenza cDNA synthesis might reflect the unique conformation of vRNA, which forms a panhandle-like structure through intramolecular pairing between 3' and 5' termini.
Phosphorylation of the 5' terminus inactivated oligo (dT) to a level of a few percent of the 5'-hydroxyl oligo (dT) in priming poly (A)-dependent poly(dT) synthesis, implying that the 5' phosphate interferes with the priming activity of oligonucleotides by a yet unidentified mechanism. However, the observed interference decreased concomitantly with increase of primer chain length.

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A chloroplast-type ferredoxin was purified from a fern, Gleichenia japonica, and its amino acid sequence was determined. The conventional method for soft leaves proved to be unsuitable for the extraction of ferredoxin from G. japonica, but a good yield was obtained by blending the leaves in cold acetone. The analysis of 8 tryptic peptides of Cm-ferredoxin gave the complete amino acid sequence. The molecule consisted of a single polypeptide chain of 95 amino acid residues and lacked tryptophan. Relatively high contents of phenylalanine and arginine were noted, some of which had unique locations in comparison with other ferredoxins. G. japonica ferredoxin did not show a close sequence homology with the ferredoxins from horsetails, which, like ferns, belong to Pteridophyta, or with those from plants of different taxonomical groups. The fern ferredoxins were suggested to form a unique group in the chloroplast-type ferredoxins.

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The effect of salts on the dimerization and the catalytic activity of dimeric α-chymotrypsin (α-CT) was investigated. The observed effect was mainly related to the anionic constituent of the salt, and its order depended on the salt concentration. The order of effect of anions on several parameters at [salt]<0.02M was SO₄^2-, ClO₄^-, NO₃^-, Br^-, Cl^-, which reflected the electrostatic interaction between the anion and the positively charged surface of α-CT. On the other hand, the anion dependence at moderate salt concentrations (0.1-0.2M) followed the Hofmeister series, SO₄^2-, Cl^-, Br^-, NO_3-, ClO₄^- which was related to the direct and indirect interactions between the anion and non-charged groups of the protein. The anion order for the dimerization constant of this enzyme, however, was the complete reverse of that generally observed in the aggregation of proteins at high salt concentration (>1M). This result was accountable for in terms of a specific interaction in the formation of the dimeric enzyme (probably that between the active site of one monomer and Tyr-146 of the other).

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After exhaustive digestion of bovine corneas with a protease, keratan sulfate fractions of different chain length were obtained by ethanol fractionation on a cellulose column, followed by ion-exchange column chromatography. Compositional analysis of these fractions showed that aspartic acid is the predominant amino acid and that the keratan sulfate in each fraction contains aspartic acid and mannose in the molar ratio of 1:3. Molecular weights of these fractions, estimated by gel chromatography, were close to the calculated values based on the molar ratios of the components to three moles of mannose. The results strongly suggested that the keratan sulfate of bovine cornea contains three mannose residues per chain, as an integral component of the linkage region to protein.

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The nucleotide sequence of 5 S RNA from rat liver ribosomes was determined by post-labeling procedures. The determined sequence coincided with the published human 5 S RNA sequence.

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The structures of the gramicidin S synthetase 2 s (GS 2, heavy enzyme) from a wild strain and mutant strains of Bacillus brevis have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS).
The GS 2 s used were obtained from a wild strain and group IV of mutant strains (BII-3, BI-3, BI-9) which lacked one specific amino acid activating activity. SDS polyacrylamide gel electrophoresis of GS 2 bound to a radioactive substrate showed that: first, in the case of the wild enzyme, the radioactivity of the substrate amino acid was detected only in the polypeptide with a molecular weight of about 280, 000, regardless of the amino acid species used as substrate; secondly, in the case of the mutant enzyme, the radioactivity of the amino acid which could be activated by the enzyme was also associated with the protein band with a molecular weight of about 280, 000.
Regardless of the enzyme species tested, a pantothenic acid residue was also present in the protein band with a molecular weight of about 280, 000.
These results suggest that GS 2 is a multifunctional one polypeptide enzyme and the mutant-type GS 2 s from BII-3 (proline-lacking), BI-3 (valine-lacking), and BI-9 (leucine-lacking) are also multifunctional enzymes having molecular weights identical to that of the wild-type enzyme.

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Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K 12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity by rifamycin SV, when Mn²⁺ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K 12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells.
The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn²⁺, Cu²⁺, and Co²⁺. The most effective metal ion was Mn²⁺. In the presence of Mn²⁺, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn²⁺-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.

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The formation of Mn²⁺- or Cu²⁺-rifamycin SV complex and the semiquinone radical of rifamycin SV was studied by electron spin resonance spectroscopy. The results indicate that: (1) the complexes Mn²⁺-rifamycin SV (g//=5.137, g⊥=2.029) and Cu²⁺-rifamycin SV (g//=2.265, g⊥=2.087, A//=177×10^-4cm^-1) were formed; (2) in Cu²⁺-rifamycin SV, Cu²⁺ was of square-planar type and was ligated to oxygen atoms; (3) the semiquinone radical of rifamycin SV (g=2.0031) was formed only under aerobic conditions and its formation was inhibited by superoxide dismutase.
The formation of hydroxyl radicals during the autoxidation of rifamycin SV was detected by the spin trapping method using N-t-butyl-α-phenylnitrone. Superoxide dismutase, mannitol and EDTA inhibited the development of the signal of the spin adduct, indicating that the hydroxyl radical is formed via the Fenton-type reaction.

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In intact human platelets activated by thrombin, diacylglycerol is produced with the concomitant disappearance of phosphatidylinositol (PI). This reaction is associated with phosphorylation of a protein having a molecular weight of about 40, 000 (40 K protein) and serotonin release. All the reactions are inhibited in a parallel manner by incubation of platelets with either dibutyryl cyclic AMP or 8-bromocyclic GMP, prior to the stimulation by thrombin. The inhibition of these reactions is inversely related to phosphorylation of another group of platelet proteins. Since Ca²⁺-activated, phospholipid-dependent protein kinase (C-Kinase) is activated by diacylglycerol and is responsible for 40 K protein phosphorylation (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., & Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317), the results suggest that in platelets both cyclic AMP and cyclic GMP may serve as inhibitors of C-Kinase by counteracting the receptor-linked PI breakdown probably through the actions of cyclic nucleotide-dependent protein kinases.

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Direct measurement has been made of the reaction rate of binding of a bivalent antibody and fluorescent haptens, which were covalently bound on a model membrane surface, by a method of stopped-flow fluorometry. The result was interpreted as indicating that the reaction takes place in two steps: (i) binding of a hapten with one of the two antigen-combining sites of an antibody molecule, and (ii) binding of another hapten with the other site of the antibody molecule in question. The rate of the second step was found to depend on the fluidity of the membrane.
We have prepared a lipid hapten (I) with a fluorescent group on its head:
H₂C-O-CO-(CH₂)₁₄CH₃
CH₃(CH₂)₁₄CO-O-CH O
H₂C-O-P-O-(CH₂)₂ NH-CH₂-CO-NH-(CH₂)₂-NH-_??_-
O-
SO₃

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An actin-regulatory protein, 88 K protein-actin complex (88 K-actin complex) isolated from porcine brain, increases the rate of actin polymerization as measured by viscometry which gives rise to shearing stresses, In contrast, when measured by the optical absorption method without solvent flow, the complex does not promote actin polymerization but inhibits both the initial rate and final extent of polymerization. These results suggest that shearing stresses are capable of modulating association of actin with the 88 K-actin complex.

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In the presence of a spin trapping reagent, N-tert-butyl-α-phenylnitrone, anaerobic incubation of halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane) with a reconstituted cytochrome P-450 enzyme system of rabbit liver microsomes exhibited an electron spin resonance spectrum containing signals of a nitroxide radical, and all components of the reconstituted system were necessary to produce the nitroxide radical. Formation of 2-chloro-1, 1, 1-trifluoroethane, the product of anaerobic dehalogenation of halothane by the reconstituted system, was inhibited by N-tert-butyl-α-phenylnitrone. These results indicate that a radical intermediate of halothane is produced during the anaerobic dehalogenation reaction, and that the radical is trapped by N-tert-butyl-α-phenylnitrone to form a N-tert-butyl-α-phenylnitrone radical adduct.

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Kidney prorenin was converted to a form of active renin by kidney cathepsin B isozymes. The three isozymes showed similar catalytic behavior for prorenin. The optimal pH for the activation was in the range of 4.0-5.0 and the reaction was completely inhibited by leupeptin. The molecular weight and the isoelectric point of the activated prorenin were 40, 000 and pH 4.9. As a minor product, an activated prorenin having an isoelectric point of 5.2 was also produced.

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Single muscle fibers were isolated from soleus and extensor digitorum longus muscle of adult rats. The muscle fiber type of single fibers was determined physiologically by the skinned fiber method according to the sensitivity to strontium (Sr) ions. The fiber type of single fibers was contrasted to the pattern of myosin light chains analyzed by one and two dimensional gel-electrophoreses. All the type 2 fibers isolated from soleus muscle contained both fast and slow types of myosin light chains.

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