Low molecular weight RNAs were isolatedfrom nuclei of the cellular slime moldDictyostelium discoideum AX-3. Analysis of the RNAs by polyacrylamide gel electrophoresis showedthat the vegetative cell nuclei contained, besides tRNA (Dd1), 5 S RNA (Dd4), and5.8 S RNA (Dd7), at least 7 small RNA species (Dd3, Dd5, Dd6, Dd8, Dd9, Dd10, Dd11) of 4 S to 8 S as major components andthat the 7 small RNAs were localizedmainly in the nucleus andhadno poly (A) sequence. These nuclear RNA species were metabolically stable, as shown by a chase experiment. Dd6, Dd8, andDd9 hadsimilar gel electrophoretic mobilities to those of the small nuclear RNA species U 1, U 2, andU 3, respectively, of rat liver. Analysis of the 5' -terminus of these RNA molecules with tobacco acidphosphodiesterase suggestedthat Dd6, Dd8, andDd9 each contain a cap. Sequence analysis of the 3' -endlabeledDd9 RNA showedthat the 3' -terminal region sequencedhadsequence homology with that of rat Novikoff hepatoma U 3 RNA. These results indicate that Dictyostelium nuclei contain a set of small nuclear RNA species which is structurally similar to that in mammalian cells. No qualitative differences were detectedbetween the small nuclear RNA species of vegetative andearly differentiating cells.

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Superprecipitation of reconstituted actomyosin composed of smooth muscle myosin, skeletal muscle actin and smooth muscle native tropomyosin was studied. When the actomyosin solution was preincubated in the presence of ATP and the absence of Cat⁺, or in the relaxed state, superprecipitation was markedly suppressed. The extent of suppression was correlated with the inhibition of the phosphorylation of the 20, 000-dalton light chain of smooth muscle myosin. This is consistent with the theory that the interaction of smooth muscle actomyosin is regulated by the phosphorylation of myosin light chain through a system of myosin light chain kinase and phosphatase. However, further studies showed that the myosin light chain kinase and phosphatase system could not explain the present suppression of superprecipitation, even if a cyclic AMP-dependent protein kinase system was also involved. A new regulatory factor should be taken into account in the regulation of smooth muscle actomyosin interaction.

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1. Difference UV absorption spectra of chicken breast g₁ induced by magnesium and calcium ions were compared with those of rabbit skeletal alkali light chains. The value of Δε due to the burial of Tyr 180 of domain 4 was about one-fourth of that of rabbit alkali light chains. Regions other than domain 4 appear to affect the environment of Tyr 180, since the first-order structure of domain 4 of chicken g₁ is present in rabbit g₁ without significant alteration (Matsuda, G. et al. (1981) FEBS Lett. 126, 111). Movement of a phenylalanyl residue was suggested to be larger in chicken g₁ than rabbit g₁.
2. CD spectra of alkali light chains were measured in the wavelength region down from 260nm. Divalent metal ions increased the helix content of alkali light chains about 2%. CB₁ peptide of rabbit skeletal g₁ containing domain 1 showed a slight change of CD spectrum in the presence of divalent ions. These results suggest that domain 1 of alkali light chains binds divalent ions.
3. Binding of calcium ions to rabbit skeletal g₁ was directly measured by means of a calcium ion selectrode. More than 3 mol of calcium was bound per mol of g₁. The concentration of free calcium ions required to give a half-maximal binding was 5mM assuming that the maximum binding number is 4 mol of calcium per mol of g₁. 4. Nitration of tyrosyl residues in rabbit skeletal g₁ may slightly affect the affinity for divalent metal ions and the backbone structure of the polypeptide chain of g₁.

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A post-proline cleaving enzyme [post-proline endopeptidase: EC 3.4.21.26] was purified from lamb brain by a series of column chromatographies on DEAE-Se-phadex, hydroxyapatite and Sephadex G-150. The purified enzyme appeared homogeneous on disc gel and sodium dodecyl sulfate (SDS) gel electrophoreses. The enzyme was most active at pH 7.0 with carbobenzoxy-Gly-Pro-β-naphthylamide (Z-Gly-Pro-2-NNap) as a substrate and catalyzed the hydrolysis of oxytocin, vaso-pressin, thyrotropin releasing hormone (TRH), substance P, luteinizing hormone releasing hormone (LH-RH), and angiotensin at the carboxyl side of their proline residues, except for the Pro²-Lys³ bond in substance P. From the results of subsite mapping using synthetic peptides, five subsites, S₃ to S₂', for substrate interaction with the enzyme were deduced to be present, and high stereospecificity was observed at S₂, S₁, and S₁'. The isoelectric point of the enzyme was at pH 4.9, and the molecular weights estimated by gel filtration and SDS gel electrophoresis were 74, 000 and 77, 000, respectively. The enzyme was markedly inhibited by diisopropylphosphoro fluoridate (DFP), carbobenzoxy-Gly-Pro-chloromethyl ketone (Z-Gly-Pro-CH₂Cl), p-chloromercuribenzoate (PCMB), Hg²⁺, and Cu²⁺ ions. These enzymatic and protein chemical properties of post-proline cleaving enzyme from lamb brain closely resemble those of the lamb kidney enzyme, except for the molecular weight. In the present work, however, we decided that the molecular weight of the enzyme from lamb kidney was also 74, 000, which is different from that reported previously (J. Biol. Chem. 251, 7593 (1976)) but is in accord with the value of post-proline cleaving enzyme from lamb brain.

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Bindings of calcium to lysozyme and its derivatives were studied by UV difference spectroscopy at various pH's. The binding constant was ca. 40M^-1 at around neutral pH. The binding caused proton release from lysozyme and did not inhibit the binding of tri-N-acetylglucosamine to lysozyme. In the presence of 0.2M Ca²⁺lysozyme showed 26% of the activity of the free enzyme toward hexa-N-acetylglu-cosamine but the cleavage pattern was similar to that of the free enzyme. Thus, calcium was predicted to bind near the catalytic carboxyls to cause inhibition of lysozyme activity. It was found from the results of protease digestion that calcium binding shifted the native-denatured transition in lysozyme toward the native state.

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Following the intraperitoneal administration of [³H] tryptophan and [³⁵S] methionine to partially hepatectomized rats, nuclei and chromatin were prepared from the liver. Three protein fractions were prepared from the chromatin by differential salt extractions. The 0.35M NaCl-soluble (0.35 MS) fraction was rich in non-histone chromatin proteins (NHCPs) and the core histones, whereas the 0.35M NaCl-precipitate (2 MS) fraction was composed of the five classes of major histones and two low molecular-weight NHCPs. The 2M NaCl-precipitate (2 MP) fraction was mainly composed of high molecular-weight NHCPs. The incorporation levels were higher in the 0.35 MS and 2 MP fractions than in the 2 MS fraction. In particular, the 2 MP fraction revealed the highest incorporations 14 h after partial hepatectomy (prereplicative phase). The ratio of ³H/³⁵S was also always higher in the 0.35 MS and 2 MP fractions than in the 2 MS fraction.
Two chromatin subtractions, 2mM Mg²⁺-soluble (S 2) and insoluble (P 2) fractions, were prepared after the digestion of chromatin with DNase II. The S 2 fraction was mainly composed of the core histones and high molecular-weight NHCPs, whereas the P 2 fraction was enriched in the five classes of major histones and low molecular-weight NHCPs. The incorporation levels of the above amino acids were always higher in S 2 than P 2. The ratio of ³H/³⁵S was also higher in S 2 than P 2 14 h after partial hepatectomy. In addition, the incorporations into 0.35 MS, 2 MP, and S 2 fractions were considerable even at 0 time. From these findings, it is evident that the high molecular-weight NHCPs were synthesized preferentially in the prereplicative phase, and showed a rapid turnover in normal liver.
On the other hand, the incorporation of [¹⁴C]leucine into the histone fraction was enhanced during the DNA replication. However, the incorporation levels of the 2 MS fraction were elevated at 14 h and further elevated at 20 h after partialhepatectomy although the levels were lower than in the other two-protein fractions. These results suggest histone synthesis before the onset of DNA replication. In this connection, the quantitative changes in low molecularweight NHCPs, HMG and A 24 proteins were examined by electrophoretic analysis. One of HMG proteins adjacent to H1 histone and A 24 protein increased in the regenerating livers whereas two other HMG proteins, probably HMG 14 and 17, decreased drastically from the prereplicative to the replicative phase.

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The kinetics of the hydrogen-deuterium exchange reaction in sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle was followed by infrared absorption measurements. The exchange rate in SR was much lower than that in the soluble proteins reported so far. When adenylyl-imidophosphate (AMPPNP, an ATP analog) was present, the exchange rate was lower than that in free SR and it was the lowest when ADP was present. The effect of the nucleotides on the exchange rate reflects the conformational change of the Ca²⁺, Mg²⁺-ATPase of SR membranes on binding the nucleotides. The structure of the Ca²⁺ Mg²⁺-ATPase is more restricted in the following order: SR+ADP>SR+AMPPNP>free SR.

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Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D→A→E→B) in Streptomyces rosa var. notoensis, was purified from the crude extract of the strain by ammo-nium sulfate fractionation and column chromatography on DEAF-cellulose, Sephadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a flavoprotein which contains FAD as a prosthetic group and has a molecular weight of 68, 000 daltons. It catalyzed the reductive transformation of nanaomycin D to nanaomycin A in the presence of NADH under anaerobic conditions. The K_m values were 250 μM for nanaomycin D and 62 μM for NADH. The enzyme was inhibited by 1mM Cul²⁺ ion and by NADH at concentrations over 50 μM. The optimal pH was 5.0 and the optimal temperature was 37°C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and frenolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4α, 10α-epoxynanaomycin D were not converted.

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Phospholipase A₂ from the venom of Trimeresurus flavoviridis (Habu snake) on treatment with cyanogen bromide is split into <Glu-Gly-Leu-Trp-Gln-Phe-Asn-Hse>, which is derived from the N-terminal moiety and is designated as S-peptide, and the remaining large peptide, which is designated as L-peptide. It should be stressed that the N-terminal residue is pyroglutamyl, unlike other phospholipases A₂. The L-peptide alone was about 6% as active as the parent molecule. It occurs in dimeric form, like the parent molecule. When L-peptide was mixed with increasing amounts of S-peptide, the activity increased in a hyperbolic manner, indicating the formation of an ordered complex between L-peptide and S-peptide. The dissociation constant of the complex was 2.1×10^-7M and its specific activity was 2.8 times that of L-peptide.

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The abilities of cathepsin B purified from liver to degrade purified myofibrillar proteins, myosin, actin, troponin, tropomyosin, and α-actinin from rabbit skeletal muscle were studied. The amino acids or peptides liberated from these proteins by cathepsin B were determined quantitatively by fluorometry with o-phthalaldehyde, and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At a molar ratio of cathepsin B to substrate of 1:100, the order of susceptibilities was myosin_??_troponin>tropomyosin_??_actin. α-Actinin was not degraded. Myosin heavy chain was degraded to several fragments with molecular weights of 175, 000, 170, 000, 160, 000, and 145, 000, whereas the light chains were scarcely degraded. Cathepsin B degraded troponin-T rapidly, and troponin-I more slowly, but did not degrade troponin-C. Troponin-T and troponin-I were degraded to three fragments with molecular weights of 30, 000, 18, 000, and 12, 800. Tropomyosin was degraded slightly and its product had a molecular weight of 32, 000. Actin was also degraded only slowly, and no liberated product could be detected.
Morphological changes in myofibrils prepared from glycerinated psoas muscle of rabbit during incubation with cathepsin B were observed. Three notable phenomena were observed: 1) disappearance of the Z-band in the early stage of incu-bation, 2) disappearance of the M-line following loss of the Z-band, and 3) decrease in the density of the A-band after swelling of the myofibrils.

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The cytosolic and mitochondrial forms of serine hydroxymethyltransferase [EC 2. 1. 2. 1] were purified to homogeneity from a whole homogenate of rat liver without the prior separation of mitochondria. The molecular weight of the cytosolic enzyme was 230, 000, and that of the mitochondrial enzyme was 200, 000. Each of the isozymes contained 4 mol of pyridoxal 5' -phosphate/mol. Tryptic peptide analyses of the NaBH₄-reduced and carboxymethylated isozymes showed that each contained a single peptide containing phosphopyridoxyllysine. The numbers of peptides obtained were about one-fourth of those expected from their contents of lysine plus arginine residues. These findings together with the identity of subunit molecular weight indicate that each of the isozymes is composed of 4 identical polypeptide chains. The isoelectric pH values of the cytosolic and mitochondrial enzymes were 4.95 and 5.30, respectively. Other differences between the isozymes include the amino acid composition, stability of the apoenzyme, reactivity toward L-allothreonine, and immunochemical properties.

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The mechanism of an increase in cytochrome c oxidase [EC 1. 9. 3. 1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and anti-body to the purified enzyme.
1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide.
2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme α.
3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices.
4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [³H] leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V.
The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.

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A protein, which was immunoreactive to antibody against cytochrome c oxidase, as found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner mem-brane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme α. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57, 000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.

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The sensitivity of a fluorescence labeling method ((1979) J. Biochem. 85, 989-994; 995-1002) for structure analyses of asparagine-linked sugar moieties of glycoproteins was increased by using HPLC with a fluorescence detector. Sugar moieties were separated from polypeptide portions by hydrazinolysis. Free amino groups thus exposed were acetylated and the reducing ends of sugar chains were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the use of sodium cyanoborohydride. The pyridylamino derivatives were purified on a Dowex 1 column to eliminate undesired substances. The separation and identification of the pyridylamino derivatives were carried out by HPLC with a column of C 18 reversed phase or gel permeation phase. As little as 0.1 pmol of pyridylamino derivatives can be detected. Ten microgram of Taka-amylase A was easily detected by this system. The method was also applied to some other glycoproteins.

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It was found that the injection of the extract of Amanita virosa mushroom into mice resulted in the inhibition of long chain fatty acid oxidation by liver mitochondria. The inhibition principle was identified as a-amanitin. The inhibition was observed several hours after the injection of α-amanitin at the level of about 0.2mg/kg, which corresponded to LD₅₀. Activities of carnitine acyltransferases, acyl-CoA dehydro-genases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase remained constant. Using fatty acids with various carbon chain lengths as substrates, the oxidation of long chain fatty acids was markedly inhibited with accompanying disappearance of the carnitine-dependent component. The decreases in the oxidation of medium and short chain fatty acids were less marked. It is suggested, therefore, that the site of the inhibition is the transport of long chain acylcarnitines. Peroxisomal β-oxidation was not affected by the α-amanitin treatment.

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We have investigated the nature of the reactive site in human C4, which shows covalent binding with alkylamines, heat-induced proteolysis, and hemolytic activity.
Incubation of C4 with [¹⁴C] methylamine resulted in the irreversible incorporation of 1 mol of methylamine into the α chain of C4. The amine incorporation was a peculiar feature of native C4 and was not observed with hemolytically inactive C4b. Incorporation of methylamine interfered with the activation of C4 by C_??_s. [¹⁴C] Methylamine-binding C4, like C4b, was susceptible to proteolytic digestion by a serum protease, C3b/C4b inactivator (C3b/C4bINA) and yielded large C4c (Mr=150, 000) and small radioactive C4d (Mr=45, 000) fragments.
As observed with C3 and α₂-macroglobulin, heat-induced proteolysis of the α chain of C4 proceeded to yield two α chain fragments of 60, 000 and 40, 000 dalton. The N-termini of the 60, 000 and 40, 000 dalton fragments were identified as pyro-glutamic acid and aspartic acid (asparagine), respectively. Since the N-terminusof the α chain was aspartic acid, the 40, 000 dalton fragment appeared to be derived from the N-terminal end of the α chain. Thus, it appeared likely that an active acyl residue, probably an activated glutamyl residue, was located at 40, 000 dalton from the N-terminal end of the α chain.
Native C4 contained a single thiol residue and did not yield any additional thiol residue upon incorporation of methylamine. This result excludes the possi-bility that, by analogy with C3 and α₂-macroglobulin, a putative internal thiolester linkage might also be responsible for activation of the acyl residue in C4.
The cleavage of C4 with 2-nitro-5-thiocyanobenzoic acid (NTCB) and the determination of the N-termini of two α chain fragments revealed that the thiol residue was located at 40, 000 daltons from the N-terminal end of the α chain. This result suggested that the thiol residue might be located close to the activated acyl residue in the α chain. Treatment of C4 with thiol reagents did not result in loss of the hemolytic activity, indicating that the thiol residue was not responsible for the hemolytic activity of C4.

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Pyroglutamyl aminopeptidase [pyrrolidone-carboxylate peptidase: EC 3. 4. 11. 8] from Bacillus amyloliquefaciens was inactivated rapidly and irreversibly by N^α-carbobenzoxy-L-pyroglutamyl chloromethyl ketone (Z-PGCK). The second-order rate constant of the inactivation was 1.1 × 10⁵ M^-1•S^-1, a value which is comparable to that of the clostripain-TLCK reaction. The D-isomer of this chloromethyl ketone derivative was almost inert toward the enzyme under the same conditions. The inactivation reaction was prevented by the presence of a poor substrate, pyroglutamyl-valine.The PCMB-inactivated enzyme, that was reversibly reactivated by 2-mercaptoethanol, failed to react with Z-PGCK. These results suggest that this chloromethyl ketone derivative reacts as an affinity label, presumably with the active site cysteinyl residue of the enzyme, as was reported for L-pyroglutamyl chloromethyl ketone.

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The pantothenic acid content of gramicidin S synthetase 2 (GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin Slacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1 (GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.

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The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16 S and 23 S rRNAs have almost identical affinities to AO. At 1mM Mg²⁺, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30 S subunit in comparison with free 16 S rRNA. In the 50 S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23 S rRNA. Mg²⁺ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg²⁺ in active ribosomes than in rRNAs. “Tight couple” 70 S particles are less accessible for stacking binding than free subunits. The 30 S subunits that have irreversibly lost the capability for 70 S formation under low Mg²⁺ conditions have an affinity to AO that is very different from that of active 30 S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70 S and 30 S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50 S subunits reversibly form aggregates.

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An affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4 B, was prepared by the periodate oxidation of AMP followed by coupling and condensation with amino-hexyl-Sepharose 4 B. RNase U², a purine-specific RNase, was specifically bound to this adsorbent at pH 4.5 and eluted critically at pH 5.9 in the presence of 1 M NaCl, corresponding to the pH dependence of the binding of 2'-AMP to RNase U₂. By using this affinity chromatography as a main tool, a simplified and effective purification method for RNase U₂ was established with a high yield of 58%. Another form of RNase U₂ with low specific activity, named RNase U₂-B, was eluted at a slightly higher pH from this adsorbent. RNase U₂-B was indistinguishable from the original enzyme (RNase U₂-A) in base specificity, affinity for ApA, molecular weight and amino acid composition, but was clearly different in specific activity, molecular activity for ApA, isoelectric point and conformation of molecule.
This affinity adsorbent is also effective for the detection or isolation of small amounts of base-specific RNases in crude cell extract.

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RNase T₂ bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4 B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3') -AMP (1mM) and NaC1 (>1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T₂, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36, 000 was identical with RNase T₂ in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80, 000 as high molecular weight RNase T₂ (RNase T₂-L). RNase T₂-L was still heterogeneous and was separated into five fractions, RNases T₂-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T₂ and the heterogeneities were due to the carbohydrate content, mainly galactose content.

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Prorennin, i.e. the zymogen of rennin, was extracted from fresh calf stomach and purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. The purified prorennin was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. A specific antibody to the purified prorennin was induced in rabbits. In a double diffusion test and on immunoelectrophoresis the antibody gave a single precipitin line when run against crude and pure zymogen preparations. In the double diffusion test, the antibody also reacted with a purified rennin but not with gastric proteases (pepsin and pepsinogen) or other proteases (trypsin and Mucor rennin). When the antibody was tested against prorennin and rennin in the double diffusion test, a spur was formed at the junction of the precipitin lines against prorennin and rennin. This result confirms that the two proteins share an antigenically common structure in their molecules and that the activation segment of prorennin contains at least one antigenic determinant.

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Six basic isoenzymes, E1 to E6, of horseradish peroxidase were isolated and purified by CM-Sephadex column chromatography, and were then crystallized. The crystalline basic isoenzymes had a lower molecular weights than the neutral isoenzyme C, but the molecular weights of their protein moieties were similar to that of the latter. The lower molecular weights of the basic isoenzymes were thus essentially due to smaller contents of carbohydrate. Of the six isoenzymes, four isoenzymes, E3 to E6, had extremely high pI values of over 12 and appreciably low contents of carbohydrate, 0.8 to 4.2%, whereas the isoenzymes E1 and E2 contained relatively large amounts of carbohydrates, 12.8 and 14.1%, respectively, and had lower pI values, 10.6. The amino acid compositions of the basic isoenzymes were different from one another, and the differences suggested that the isoenzymes were allelic. The relatedness of the amino acid compositions of these isoenzymes is discussed and the compositions are compared with those of other peroxidases. Finally, one of the six isoenzymes, E4, was obtained as large single crystals suitable for X-ray structural analysis, and the preliminary crystallographic data are presented.

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Chickens of a dystrophic line are characterized by the persistence of a high rate of amino acid incorporation into actin until adolescence and by the augmentation of amino acid incorporation into α- and 1O S-actinin in adulthood.

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Human high molecular weight (HMW) kininogen is a single chain with a molecular weight of 120, 000 and is cleaved by plasma kallikrein sequentially into a nicked kininogen, an intermediate kinin-free protein (KFP-I), and a stable KFP-II. Here we report a study into the process of cleavage of human HMW kininogen by human salivary kallikrein.
On incubation with salivary kallikrein, HMW kininogen was first converted into a nicked kininogen composed of disulfide-linked chains of 62, 000 and 56, 000 daltons. Subsequently, the nicked kininogen was cleaved into kinin and a KFP, which was apparently of equal size to the nicked kininogen, that is, KFP-I. In contrast to plasma kallikrein, salivary kallikrein did not cleave KFP-I into KFP-II.
The two chains were separated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I. The N-termini of HMW kininogen and the 62, 000-daltons chain were found to be pyroglutamyl-isoleucyl, while that of the 56, 000-daltons chain was found to be serine.
These results indicate that the sequence of the two chains and kinin in human HMW kininogen is 62, 000-daltons chain-kinin-56, 000-daltons chain from the N-terminal end of HMW kininogen.
Possible processes of cleavage of human HMW kininogen by human plasma and salivary kallikreins are also discussed.

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Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89, 000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154, 000 and 149, 000, respectively. They each consist of 4 polypeptide chains of identical size.
Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.

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In the presence of excess amounts of various monosaccharides as acceptors, Bacillus subtilis levansucrase synthesized various heterooligosaccharides as a result of transfer of the fructosyl residue from sucrose. The saccharides produced in the presence of D-glucose, D-mannose, and D-xylose were all non-reducing disaccharides and were identified as β-D-fructofuranosyl-α-D-glucopyranoside (sucrose), β-D-fructo-furanosyl-α-D-mannopyranoside (mannosucrose), and β-D-fructofuranosyl-α-D-xylopyranoside (xylsucrose), respectively. The saccharides produced in the presence of D-galactose were also non-reducing, but consisted of di-, tri-, and tetrasaccharides. The di- and trisaccharides were galsucrose and 6^F-β-D-fructofuranosylgalsucrose.
The saccharide synthesized in the presence of L-arabinose was a reducing saccharide and was determined to be 4-O-β-D-fructofuranosyl-L-arabinose. In the presence of D-fructose, several reducing levan oligomers, levanbiose, levantiose, levantetraose, etc., were produced, which were not observed in the synthesis of levan from sucrose alone.

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The time-courses of substrate consumption and product formation in the lysozymecatalyzed reaction were determined with (GlcNAc)₄ and (GlcNAc)₅ as substrate to accumulate data suitable for the estimation of rate constants by numerical analysis. The lysozyme-catalyzed reactions were followed by TLC or HPLC. (GlcNAc)₄ decomposed apparently to small oligosaccharides within 5 h, and (GlcNAc)₅ decomposed within 15 min at pH 5.0 and 50°C. The temperaturedependence of the rate of disappearance of the initial substrate showed a different profile from that observed with glycol chitin as substrate by the reducing power method. The order (or distribution) of the amount of product formed from (GlcNAc)₅ in the reaction time-course determined by TLC differed from that determined by HPLC. The relative error in HPLC was much less than that in TLC, and the time-course determined by HPLC was thought to be of sufficient accuracy for the estimation of rate constants by computer analysis.

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On RPC-5 column chromatography, the main valine acceptor activity of tRNA (tRNA₂^Val) from rat ascites hepatoma cells was eluted later than that of normal rat liver tRNA (tRNA₁^Val). The tRNA₂^Val was aminoacylated by E. coli aminoacyl-tRNA synthetase, while tRNA₁^Val from normal rat liver was not. Rat fetal liver tRNA^Val was also aminoacylated by E. coli aminoacyl-tRNA synthetase. tRNA₁^Val (rat liver) and tRNA₂^Val (ascites hepatoma) were each purified to a homogeneous state by RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis, and their sequences were determined by post-labeling techniques. Ascites hepatoma tRNA₂^Val differed from rat liver tRNA₁^Val in that Gm₁₈, C₃₂ and an unknown modified nucleoside, N₃₄, in the latter tRNA were mostly replaced by G, Cm, and inosine, respectively. In addition, 3'-terminal adenosine was not present in tRNA₁^Val (normal rat liver), but was in tRNA₂^Val (ascites hepatoma). Other modifications and the primary structures of the two tRNA^Vals were found to be the same. Thus it was concluded that the new isoacceptor species of tRNA_Val in ascites hepatoma cells is due to a change of post-transcriptional modification, not to a change of tRNA transcription. The unique feature of the change of post-transcriptional modification in tRNA₂^Val (ascites hepatoma) is that both hypo- and hyper-modification take place simultaneously in the tRNA molecule depending the locations of nucleotide residues.

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The reactivity of the sulfhydryl groups in colonic myosin B to N-ethylmaleimide (NEM) was studied under various conditions. A higher concentration of NEM was required to alter the ATPase activity as compared with skeletal myosin B. A chemical modification of colonic myosin B at a low ionic strength brought about the marked inhibition of EDTA-ATPase and 30-40% activation of Mg²⁺-ATPase, but had little effect on Cat²⁺-ATPase. The same results were obtained with NEM modification at a high ionicstrength. The presence of 10mM MgCl₂ in the reaction medium for NEM modification caused the activation of Cat²⁺-ATPase and remarkable inhibition of EDTA-ATPase, suggesting that SH₁, is here reactive to NEM. NEM modification of colonic myosin B in the presence of ATP resulted in the inhibition of not only EDTA-ATPase but also Ca²⁺- and Mg²⁺-ATPases although the degree of inhibition was different. Addition of 2.0M urea to the modification medium caused the simultaneous inhibition of EDTA- and Ca²⁺-ATPase but little change in Mg²⁺-ATPase. Based on these results, the presence of three specific sulfhydryl groups, SH_a, SH₁, and SH₂, is discussed.

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Upon Cs₂SO₄ density-gradient equilibrium centrifugation, 60 S subunits of rat liver ribosomes were dissociated to form three bands at the densities of 1.55, 1.40, and 1.30g/ml. The bands at 1.55 and 1.30g/ml were shown to contain exclusively RNA or proteins, respectively, whereas the band at 1.40g/ml contained both RNA and a protein. The RNA in the 1.40g/ml band was identified as 5 S RNA and the protein in this band was protein L 5. An empirical calculation suggested that the 1.40g/ml band contained an equimolar 5 S RNA-protein L 5 ribonucleoprotein complex. This finding may indicate that protein L 5 is located in very close proximity to 5 S RNA in large ribosomal subunits.

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Electrophoretic analysis on sodium pyrophosphate gels of myosin light chain 1 (LC-1; 27, 000 daltons) from rabbit ventricular heart muscle revealed the presence of two distinct components named LC-1a (10%) and LC-1b (90%). After thyroxine administration, a shift toward LC-1a (45%) was observed. Canine cardiac ventricular light chain 1 (27, 000 daltons) had equal amounts of LC-1a' (50%) and LC-1b' (50%), which corresponded to LC-1a and LC-1b of rabbit heart, respectively. However, heterogeneity of light chain 2 (20, 000 daltons) was not observed. It is concluded that cardiac ventricular myosin has two heterogeneous 27, 000 daltonlight chains and that the proportion of these proteins varies according to the thyroid status and animal species.

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Incorporation of [6-³H]fucose and [1-¹⁴C]glucosamine into the lipid fraction of microvillus membrane (MVM) was studied in germ-free and conventionalized mice after intraperitoneal injection of the radioactive precursors. Incorporation of [6-³H]fucose was clearly detected in conventionalized mice but not detectable in germ-free mice. There was no difference in the incorporation of [1-¹⁴C]glucosamine between the two groups. The lipid fraction of MVM labeled with [6-³H]fucose showed a spot of slower mobility than asialo GM₁ on TLC with autoradiography and it was confirmed to be a fucolipid on abolishing the radiolabeled original spot by α-L-fucosidase treatment. These results suggest that the synthesis of the fucolipid is induced by conventionalization of germ-free mice.

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Recently, Heizmann et al. (Proc. Natl. Acad. Sci. U. S. 78, 74-77 (1981)) reported that muscle β-actinin and serum albumin of the chicken are indistinguishable by physicochemical and immunological criteria. It should, however, be stated that the protein the above authors called β-actinin was entirely different from genuine muscle β-actinin (Maruyama et al. (1977) J. Biochem. 81, 215-232). In the present study, it was experimentally shown that chicken serum albumin does not have any of the actions of β-actinin: inhibition of reassociation of F-actin fragments, retar-dation of depolymerization of F-actin, instability of F-actin, acceleration of polymerization of G-actin, and formation of Mg polymer. The role of muscle β-actinin, a heterodimer of 37, 000 and 34, 000 daltons, in the regulation of myofibrillar structure is summarized.

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A simple and rapid method for isolating tryptophan 5-monooxygenase [L-trypto-phan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating), EC 1. 14. 16. 4] was reported. The method involves adsorption on calcium phosphate gel and affinity chromatography on agarose coupled with dimethyltetrahydropteridine. Tryptophan 5-monooxygenase was purified 1, 100-fold from a rabbit brain extract to a specific activity of 15.9 nmol/min•mg, which is far higher than that of the highly purified preparation reported by Tong and Kaufman ((1975) J. Biol. Chem. 250, 4152-4158). It was also demonstrated that this method was applicable to the purification of rat brain tryptophan 5-monooxygenase.

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Isozyme patterns of S-adenosylmethionine synthetase have been determined with and without dimethylsulfoxide in liver of tumor-bearing mice. The activity of the β-form markedly increased following intraperitoneal transplantation of Ehrlich ascites tumor cells, whereas the activity of the α-form did not change so strikingly. Tumor cells were shown to have only the γ-form of the enzyme. When the ascites fluid, after removing tumor cells, was injected into a normal mouse, the activity of the β-form increased over that of control animals.

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Interaction of microtubule-associated proteins (MAPs) with actin filaments at neutral pH is inhibited by phosphorylation of MAPs. Phosphorylated MAPs are less potent than unphosphorylated ones in increasing the low-shear viscosity of actin filaments in the neutral pH range. The ability of unphosphorylated MAPs to crosslink actin filaments falls off sharply above pH 7. 5. Upon phosphorylation, the crosslinking ability of the MAPs peaks sharply between pH 6.2 and 6.3. Thus, the MAPs-actin interaction can be regulated by phosphorylation of MAPs and small changes in the physiological range of pH.

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