Male Wistar rats were fed a diet with or without di (2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator, for 2 weeks. The increases in the individual enzymes of the hepatic peroxisomal β-oxidation system after administration of DEHP were 31-to 33-fold. It was found by in vivo experiments using L-[4, 5-³H] leucine and the immunoprecipitation technique that the rates of synthesis of the enzymes were 16-to 20-fold higher and those of degradation were 1.7- to 1.9-fold lower in the DEHP group. The translation rates of these enzymes in vitro with liver RNA in the reticulocyte-lysate system were 12- to 14-fold higher in the DEHP group. Short-term kinetic labeling experiments on acyl-CoA oxidase consisting of three subunits were conducted in vivo to explore the biogenesis of peroxisomes. The label was found in the biggest subunit of the enzyme in the supernatant fraction shortly after the label injection, but was distributed to the smaller subunits later. The labeling in the smaller subunits in the peroxisomal fraction was greater than that of the supernatant. The distribution of the label among the subunits in these subcellular fractions was the same as that of the protein amounts 1 day after the label injection. This paper reports that the increase in the quantities of peroxisomal enzymes upon administration of DEHP is mainly due to the increase in their synthesis rates caused by the increase in amounts of mRNA coding for these enzymes.

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The neutral glycosphingolipids from larvae of the green-bottle fly, Lucilia caesar, were analyzed. Thin-layer chromatograms showed that the larvae contained at least seven major glycolipid components. Of them, the four components with shorter sugar chains consisting of one to four sugar units, were purified by latrobeads column chromatography. The structures were identified by partial acid hydrolysis, sequential enzymatic hydrolysis, chromium trioxide oxidation and methylation analysis as: Glcβ (1-1) Cer, Manβ (1-4) Glcβ (1-l) Cer, GIcNAcβ (1-3) Manβ (1-4) Glcβ (1-1) Cer and GalNAcβ (1-4) GlcNAcβ (1-3) Manβ (I-4) Glc (1-1) Cer.
These glycolipids altogether comprised 38.3% of the total neutral glycolipid fraction. Unlike common vertebrate glycosphingolipids, the larval ones appear to be quite unique in having the sugar structures, -GlcNAcβ (1-3) Man- and -GaINAcβ (1-4) GlcNAcβ (1-3) Man-, which are novel finding in the natural systems examined so far.
The ceramide moieties were composed of normal fatty acids (16:0-22:0) with a small amount of branched acids (16 and 18), and tetradeca- and hexadeca-4-sphingenines as the long-chain bases.

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Two forms of NADPH-adrenodoxin reductase, tightly and loosely bound forms, were found in bovine adrenocortical mitochondria. The loosely bound NADPH adrenodoxin reductase was solubilized by ultrasonication and its content was about 65 % of the total activity in bovine adrenocortical mitochondria. The tightly bound NADPH-adrenodoxin reductase could not be solubilized by mechanical sonication and required detergent treatment.
The tightly bound NADPH-adrenodoxin reductase was purified to homogeneity, judging by sodium dodecyl sulfate (SDS) gel electrophoresis, from bovine adrenocortical mitochondria using sodium cholate solubilization and chromatography on DEAE-Sepharose and 2' 5'-ADP-Sepharose columns. Its physicochemical properties were similar to those of the loosely bound form, except for its carbohydrate composition.

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1. Linear uptake of tyrosine lasted for I min in cultured B-16 mouse melanoma cells preloaded with an amino acid such as tyrosine.
2. The tyrosine uptake increased markedly on preincubating the cells with 0.1mm methionine, tyrosine, histidine or tryptophan and moderately with 1mm phenylalanine, valine, isoleucine, or leucine. The effects of the preincubation on leucine uptake were similar to those on tyrosine uptake.
3. The tyrosine uptake was Na-independent under the experimental conditions used (0.05-1.0mM); K_m 75 μM and V_max 15 nmol/min/mg protein. The methionine uptake (0.1-0.5mM) was also Na-independent (K_m 150 μM and V_max 25 nmol/min/mg protein), whereas Na-dependent uptake could contribute at 2mM methionine.
4. Inhibitions of tyrosine uptake by tryptophan, phenylalanine, leucine, isoleucine, methionine, valine, and histidine were competitive, giving Kj values of 70, 80, 100, 130, 160, 350, and 900 μM, respectively.
5. Exchange between intracellular methionine and extracellular tyrosine and vice versa was equimolar. Potencies of amino acids in stimulating tyrosine efflux were in the following order: methionine>tyrosine>histidine>tryptophan>phenylalanine>leucine>isoleucine valine.
6. The amino acid affinity of the system in the intracellular surface was suggested to be different from that in the extracellular surface.
7. The theophylline-treated cells showed a marked increase in tyrosine-uptake rate with elevated V_max and unchanged K_m

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The effects of volatile anesthetics on the properties of membrane surfaces were studied using the negatively charged fluorescent probe I-anilino-8-naphthalene sulphonate (ANS). Although the addition of the anesthetics caused no change of the fluorescence quantum yield of ANS bound to the biological membranes, there was a significant increase of the number of binding sites (n) for ANS to the membranes. This increase was also found after treating the membranes with either trypsin or neuraminidase and with vesicles of total lipids extracted from erythrocytes.
With phosphatidylcholine (PC) vesicles, the fluorescence increase by the addition of anesthetics was observed only when the vesicle contained cholesterol. The greater increase of the n value was seen in vesicles containing a higher concentration of cholesterol. When the neutral probe, N-phenyl-l-naphthylamine (NPN) was used instead of ANS, this fluorescence increase was not seen.
These results were interpreted in terms of the electrostatical change of the membrane lipid region. That is, a change of the surface potential of the membrane is possibly caused by the anesthetics through the interaction with lipid components. Cholesterol plays a critical role in this interaction.

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Salt-induced pH changes of smooth microsomes from rat liver were examined. At pHs higher than 4.9 addition of salt to a microsomal suspension induced a decrease in pH, while at pHs lower than 4.9 it induced a pH increase. The salt-induced pH changes were explained by the change in the degree of dissociation of ionizable groups of membranes due to the change of surface potential and surface pH. On comparison of the experimental data with those of calculations with the Gouy-Chapman equation, a value of 3.1±0.1×10^-3 carboxyl groups/Ų was obtained, which gives a maximal surface charge density of-1.08±0.04×10^-3 elementary charge/Ų at neutral pH and -19.2 mV surface potential at 0.15M monovalent salt.
Due to the surface charges of smooth microsomal membranes the surface pH and surface concentrations of ionic substrates become different from those in the bulk aqueous phase depending on the salt concentration. This explains part of the alt-concentration dependence of the activities of membrane enzymes in vitro. The importance of the surface concentrations of ionic substrates of enzymes of smooth microsomal membranes in vivo is also suggested.

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We studied the effect of intestinal microorganisms on the synthesis of membraneassociated glycoproteins in the upper small intestine by intraperitoneally administering L-[³H] fucose, D-[¹⁴C] glucosamine, or L-[³H] Ieucine to germ-free mice and mice exposed to microorganisms for 4 weeks (conventionalized). The incorporation of the labeled compounds into sucrase-isomaltase complex and maltase was determined by immunoprecipitating Triton X-100-solubilized microvillus membranes with their antibodies. Purified microvillus membranes from germ-free and conven-tionalized mice differed in the activities of some marker enzymes but not in the number and mobility of the components on SDS-polyacrylamide gel electrophoresis. Maximal incorporation of [³H]fucose and [¹⁴C]glucosamine into the microvillus membrane and two enzymes was reached 2-3 h post-injection in both groups, however, the amounts incorporated were larger in conventionalized mice. There was little difference in [³H] leucine incorporation into the total glycoproteins of microvillus membranes between the two groups.
Our results suggest that the introduction of microorganisms stimulates the synthesis of sugar chains of microvillus membrane-associated glycoproteins. The enhanced in vitro fucosyltransferase activity in conventionalized mice partly supports this suggestion.

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Structural changes in the Z disk were sensitively detected by measuring fragmentation indexes of myofibrils. The Ca²⁺-induced weakening of Z disks and the Z-disk removal by muscle calpain could be clearly distinguished by using muscle calpastatin, an endogenous inhibitor of muscle calpain. The Ca²⁺-induced weakening of Z disks occurred without concomitant release of a-actinin and had maxima at 10^-4M Ca²⁺ and 45°C and a minimum at pH 6.5, while the Z-disk removal by calpain had similar optima to the caseinolytic activity of calpain, at 10^-3 M Ca²⁺, 20°C and pH 7.0. The Ca²⁺-induced weakening of Z disks is therefore not due to the proteolytic action of calpain. In postmortem muscle, moreover, the Ca²⁺-induced weakening of Z disks was inferred to be predominate over calpain proteolysis, and therefore to be the major factor in the characteristic weakening of Z disks.

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Methylation and demethylation of the methyl-accepting chemotaxis protein (MCP) was studied in vitro. The in vitro MCP methylating system showed the following characteristics.
1. Multiple bands of methylated MCP were produced.
2. The reaction could be separated into two phases: the initial phase, in which the level of methylation increased as a result of multiple methylation; and the stationary phase, in which methylation and demethylation took place at the same velocity without apparent change in the level of methylation.
3. Pulse-chase analysis of the reaction showed that MCP was demethylated preferentially at the relatively highly methylated state.
4. The behavior of MCP in this system was similar to that of MCP stimulated in vivo with attractant.
Based on the above results, an autonomous control mechanism of the level of MCP methylation is discussed.

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The effects of tunicamycin (TM) on cell adhesion and on the biosynthesis of the membrane-associated glycoproteins in aggregation-competent cells of Dictyostelium discoideum were investigated. The cohesiveness due to the EDTA-stable contact site was completely inhibited in the presence of I μg/ml of TM. In contrast, when cells were treated with high concentrations of TM, the EDTA-sensitive contact site in the early developmental stage remained functional. Scanning electron microscopy showed the surface of TM-treated cells to be smoother than that of untreated cells. The membrane proteins of the aggregation-competent cells and TM-treated cells were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The results showed that several protein bands at 190 kd, 170 kd, 150 kd, 135 kd, 127 kd, 94 kd, 80 kd, 68 kd, 61 kd, and 52 kd which were observed in the untreated cells disappeared in the TM-treated cells, and protein bands at 105 kd and 70 kd were newly detected in the treated cells. Tunicamycin inhibited the incorporation of [³H] mannose or [¹⁴C] glucosamine into the trichloroacetic acid (TCA)-insoluble material of the aggregation-competent cells, while it did not affect [³H] leucine incorporation. These results suggest that an asparaginelinked carbohydrate moiety of membrane-associated glycoprotein (s) is involved in cell adhesion of aggregation-competent cells of D. discoideum.

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The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca²⁺ has been studied. When the ionic strength of an activating solution (containing Mg²⁺-ATP and Ca²⁺) was decreased by varying the KCl con-centration from 120 to 5mM at 20°C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6°C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca²⁺, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20°C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca²⁺ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6°C or at physiological ionic strength.

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A proline iminopeptidase was purified about 18, 000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purified enzyme had a molecular weight of 220, 000 by gel filtration and 55, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the native enzyme may be composed of four identical subunits. The isoelectric point was 6.2 as determined by gel electrofocusing. The pH optimum for L-proline β-naphthylamide was between pH 7.5 and 8.0, and the enzyme was stable in the pH 6.5-to-8.0 region and up to 40°C. The enzyme was specific for L-proline β-naphthylamide among various amino acid β-naphthylamides, and it also hydrolyzed L-prolylglycine and Lprolylglycylglycine. The enzyme was strongly inhibited by p-chloromercuribenzoate, 5, 5'-dithiobis (2-nitrobenzoic acid), N-ethylmaleimide, and heavy metal ions, but was not activated significantly by thiol compounds. Moreover, the enzyme was inactivated by diethyl pyrocarbonate, p-bromophenacyl bromide, and photooxidation, but was not affected by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bestatin, puromycin, or metal chelating agents. No activation of the enzyme was observed on addition of metal ions. These results suggest that the enzyme is not classifiable as a metalloenzyme, and that cysteine and histidine residues may participate in the enzyme activity.

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The adsorption of Escherichia coli phosphoenolpyruvate carboxylase [EC 4. 1. 1. 31] to butyl-, hexyl-, and octyl-Sepharose gels was investigated. The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate. At intermediate concentrations-0.1M in the case of hexyl-Sepharose-virtually no adsorption was observed. Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups. The adsorption to hexyl-Sepharose at 0.7M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators. Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors. The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids. The specific elution with L-aspartate in the presence of 0.82M ammonium sulfate could successfully be applied to purification of the enzyme. By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73%, The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis.

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Calf thymus DNA polymerases α and β [EC 2. 7. 7. 7] and terminal deoxynucleotidyl transferase [EC 2. 7. 7. 31] were analyzed on two-dimensional gel slabs. DNA polymerase β appeared as a single spot on two-dimensional gel at the position of 40, 000 daltons and pI8.0 using non-equilibrium pH gradient gel electrophoresis for the first-dimensional run. By overlapping gel slabs, it was possible to identify the distinct spot of DNA polymerase β among many polypeptide spots of a crude enzyme fraction. lOS DNA polymerase a showed two clusters of polypeptide spots on two-dimensional gel slab. One cluster was composed of three large polypeptides of 140, 000-150, 000 daltons and another was composed of four smaller polypeptides of 46, 000-50, 000 daltons. All these spots were arranged in a narrow pI range (6.5-6.8) although each spot showed a distinct pI value. Purified terminal deoxynucleotidyl transferase showed three polypeptides of 57, 000, 42, 000, and 33, 000 daltons at similar pI values (7.0-7.2). Each polypeptide consisted of plural spots which differed slightly in pI but were the same in molecular weight. These results suggest a microheterogeneity of polypeptides of terminal deoxynucleotidyl transferase as well as those of 10S DNA polymerase α.

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ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 μM, and was stimulated at concentrations between 2 μM and 10 μm. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF₁, ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.

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Tryptophan hydroxylase from mouse mastocytoma P-815 is activated by ethylenediaminetetraacetate (EDTA). The activation proceeds in a pH-, temperature-, and time-dependent manner and leads to a 30-fold higher activity at maximum. The optimal EDTA concentration is 10 μm. The activation requires solely EDTA with desalted crude enzyme solution in the absence of any cellular metabolites. There are no indications that the activation is due to proteolysis, modification of proteinbound sulfhydryl groups or other covalent modifications such as phosphorylation and methylation. In the presence of appropriate stabilizing agents, the activated state is maintained after the removal of EDTA by gel-filtration. The activation is reversible under conditions in which bound metal (s) is dissociated from the complex with EDTA. These results imply that the role of EDTA is metal chelation. A model is proposed in which the enzyme has at least two interconvertible states, the activated state and ground state, corresponding to the free and metal-bound forms, respectively. The metal is probably derived from the cell.
The assay method for tryptophan hydroxylase utilized a rapid and sensitive (5 pmol/injection) determination of 5-hydroxytryptophan by high performance liquid chromatography.

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Rat liver cytosol fraction facilitated the transfer of [³H]glucosylceramide and [³H]-galactosylceramide from donor to acceptor liposomes, which contained one of these glycolipids in phospholipid-cholesterol. Lactosylceramide transfer activity was not detectable in the cytosol fraction. On Sephadex G-75 gel filtration of the liver cytosol fraction, both the glucosylceramide and galactosylceramide transfer activities were eluted at an identical position with a K_ay value of 0.364, which corresponds to a molecular weight of 19, 500; a little lactosylceramide transfer activity was found in the effluents containing the glycolipid transfer activities. The active protein fraction obtained by Sephadex G-75 chromatography of rat liver cytosol accelerated the transfer of galactosylceramide, glucosylceramide and lactosylceramide at relative rates of 100, 79, and 31, respectively. The rates of galactosylceramide and glucosylceramide transfer facilitated by the rat liver Sephadex G-75 fraction were not affected by either galactosylceramide, glucosylceramide, or lactosylceramide con-tained in the acceptor liposomes; acceptor liposomes lacking glycolipids acted effi-ciently as acceptors in the transfer reactions. For the lactosylceramide transfer reaction, significantly lower activity was detected when the acceptor liposomes contained either galactosylceramide or glucosylceramide. The translocation of [³H]-alactosylceramide from the donor to acceptor liposomes was confirmed by TLC and fluorography of the lipids extracted from the acceptor liposomes, which were separated from the donor liposomes after the incubation.

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Two types of sarcoplasmic reticulum (SR) vesicles, i.e., normal (NSR) and heavy (HSR), were prepared by using a centrifugation technique, and further characteri-zation of the cation transport in the two types of SR vesicles was performed by measuring the choline influx. The choline influx in SR vesicles was measured by following the change in light scattering intensity using a stopped flow apparatus. Analysis of the choline influx gave the following results. (1) There exists a Ca²⁺-gated cation channel in SR vesicles other than the K⁺-selective, voltage-dependent cation channel. The Ca²⁺-gated cation channel was activated by both extravesicular Ca²⁺ and caffeine, and inhibited by procaine and N, N'-dicyclohexylcarbodiimide. (2) About 70 % of the HSR vesicles have Ca²⁺-gated cation channels, whereas only 20% of NSR vesicles have these channels. On the other hand, 60% of HSR vesicles also have the voltage-gated cation channels and 80% of NSR vesicles have these channels. On the basis of various properties of the Ca²⁺-gated cation channel, it is suggested that this channel must be the one which mediates the Ca ²⁺-induced Ca²⁺ release.

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The effects of caffeine and procaine on the Ca ²⁺-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx in SR vesicles was measured by following the change in light scattering intensity using a stopped flow apparatus. From the kinetic analysis of the rate of choline influx, the following results were obtained. (1) The rate of choline influx was enhanced when Ca²⁺ bound to the Ca²⁺-receptor site of the Ca²⁺-gated cation channel. (2) Caffeine enhanced the choline influx by increasing only the affinity of Ca²⁺ for the receptor site of the channel and thus regulated the equilibrium between open and closed states of the channel. The affinity increased about 14-fold upon caffeine binding. The dissociation constant of caffeine was 10mM. (3) In contrast, procaine itself blocked the choline influx mediated by the Ca²⁺-gated cation channel. The blockade followed a single-site titration curve with a Ca²⁺-dependent dissociation constant of 0.44mM at 2×10^-6M Ca²⁺. The Ca²⁺-dependence was explained by assuming that procaine would bind to the inhibitory site only when the channel was open. (4) Procaine also inhibited the choline influx enhanced by caffeine. The blockade could be explained on the basis of the above kinetic model.

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The mechanism and function of the Ca²⁺-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring both choline influx and Ca²⁺ efflux. The choline influx was measured by the light scattering method using astopped flow apparatus. The Ca²⁺ efflux was measured by the Millipore filtrationmethod using ⁴⁵Ca. Kinetic analysis of the rates of choline influx and Ca²⁺ efflux led to the following results.
1. The Ca²⁺-activated choline influx was dependent on the choline concentration. It increased with increase of choline concentration in a cooperative manner (n_H=2.6) and was saturable at high choline concentrations. The apparent dissociation constant for choline was 0.39M.
2. Caffeine increased the Ca ²⁺-activated choline influx according to the Michaelis Menten equation at low concentrations of choline, whereas the action of caffeine became cooperative (n_H=1.9) at higher concentrations of choline, suggesting that there is a cooperative interaction between channels.
3. The Ca²⁺-activated choline influx was inhibited competitively by Ca²⁺. One Ca²⁺ competes with 2 cholines. The inhibition constant for Ca²⁺ was 22 μM and the dissociation constant of choline was 0.28M, suggesting that the competition site is the transport site of choline.
4. Various polyvalent cations also blocked the channel. The apparent inhibition constants for the inhibitors were as follows: La³⁺ 0.01 μM, Co²⁺ 7 μM, Mn²⁺ 8 μM, Mg²⁺ 14 μM, Ni²⁺ 19 μM, Ca²⁺ 22 μM, Ba²⁺ 30 μM, and Sr²⁺ 40 μM.
5. The inhibition of Ca²⁺-activated choline influx by Mg²⁺ or Co²⁺ was competitive with respect to choline. One Mg²⁺ or Ca²⁺ competes with 2 cholines. The apparent dissociation constant of choline was 0.31M, suggesting that the competition site is also the transport site of choline.
6. An organic Ca²⁺ antagonist, nicardipine, blocked the Ca²⁺-gated cation channel with a dissociation constant of 5.4 μM. Ruthenium red and arsenazo III alsoblocked the channel with apparent dissociation constants of 0.07 μM and 16 μM, respectively.
7. The channel was also blocked by a fluorescent cyanine dye, diS-C_3-(5), and by quinine, which are potent inhibitors of the Ca^2+-activated K⁺ channel in red blood cell membrane; the apparent inhibition constants were 4.4 μM and 240 μM, respectively. However, blockers of the K⁺ channel in nerve membrane, such as TEA and 3, 4-diaminopyridine, were not effective.
8. When SR vesicles were modified with N, N'-dicyclohexylcarbodiimide (DCCD), both Ca²⁺-activated choline influx and Ca²⁺-activated Ca²⁺ release from SR vesicles were blocked. The apparent inhibition constant of DCCD was 80 μm. The blockade was prevented by Ca²⁺. The Ca²⁺ concentration required for half-maximal protection was about 1mM.
These results suggest that two cholines are simultaneously transported by one channel and there exists a positive homotropic cooperativity between some channels. In addition, from the pharmacological data, we concluded that the so-called Ca²⁺-gated cation channel is the Ca ²⁺ channel existing in sarcoplasmic reticulum.

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The binding of myosin subfragment-I (S-1) to F-actin in the absence of nucleotide was examined by the sedimentation method using 1, 5-IAEDANS-labeled S-1. We found that the binding affinity of F-actin to S-1 was dependent on the concentration of F-actin, and the binding was weaker at higher concentrations of F-actin. The apparent association constant determined from a linearly extrapolated Scatchard plot was 6.5×106M^-1 at 8.1 μM F-actin, and 1.7×10⁷M^-1 at 2.0 μM F-actin in 120mM KCl, 2mM MgCl₂ 0.1mM CaCl₂, and 20mM Tris-acetate (pH 7.6) at 20°C. Furthermore, the Scatchard plot revealed the existence of cooperativity in the binding of S-1 to F-actin. In order to obtain higher precision we developed a new method for the chromatographic determination of free S-1 in acto-S-1 solution. By this method we could determine free S-1 concentrations of the order of 10-9M easily and accurately. The above conclusion obtained by the sedimentation method was confirmed by this chromatographic method, and these effects can be well explained by considering the length distribution of F-actin. We propose an allosteric model in which both the length distribution and the polarity of F-actin are taken into consideration

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Dialysis of the peroxisomal extract, microsomal extract and cytosol fraction of rat liver against a solution containing 44mM acetate buffer, pH 4.1, and 22 % ethanol resulted in elimination of immature and enzymatically inactive catalase as insoluble precipitates, leaving mature and active catalase in solution.
These catalase molecules in the three different subcellular compartments were doubly labeled with injected radioactive amino acids, for 30 min with [³H] leucine and for 90 min with [¹⁴C] leucine. The specific radioactivities of peroxisomal catalase remained unchanged upon the dialysis described above. This was also the case with microsomal catalase, however, the values were much higher than those of peroxisomal catalase. On the other hand, cytosol catalase showed remarkably decreased radioactivities after the dialysis, which were comparable to those of peroxisomal catalase, and the immature and inactive catalase which had been removed by this treatment was estimated to be as highly radioactive as the microsomal catalase.
By affinity chromatography using anticatalase antibody both the microsomal extract and cytosol fraction were found to contain enzymatically inactive and probably immature catalase, which did not occur in peroxisomal extracts. However, when examined by SDS-polyacrylamide gel electrophoresis, the catalases in the three different subcellular fractions gave identical single bands corresponding to the monomer subunit of this enzyme protein, and no other larger molecule was detected.
It was also found that the peroxisomal mature catalase did not migrate in polyacrylamide gel electrophoresis the same as that in microsomes and in cytosol, the latter two exhibiting the same mobility.
Based on these results and others previously obtained, intracellular events in the maturation and transfer of newly synthesized catalase are discussed.

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A combination method consisting of separation by HPLC using gel permeation columns and selective detection of cholesterol or choline-containing phospholipids was applied to the identification and quantitation of subclasses of high density lipoproteins in human serum.
The frequency distribution of peaks in the elution patterns on detection of choline-containing phospholipids in a group (n=71) of normal males and females showed five maxima. Their particle sizes were determined from the relation between Stokes' diameter and elution volume to be as follows: 122.0±2.8 Å, 110.1±2.1 Å, 97.5±1.8 Å, 86.7±1.3 Å, and 76.3±1.6 Å. The two larger fractions were found to correspond to HDL₂ subclasses, i.e., HDL_2b and HDL_2a. The other three peaks were found to reflect the subclasses of HDL₃ and choline-containing phospholipids in the very high density lipoprotein fraction which sedimented at the bottom on ultracentrifugation at the density of 1.21.
Moreover, the existence of these five subclasses in the HDL fraction was confirmed by rechromatography using this combination HPLC method.
Our results for the particle sizes of HDL subclasses were confirmed by electron microscopy.

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In marine fish and crustacean liver, degradative enzymes able to convert purines to urate have been shown to be located only in the cytosol, and degradative enzymes able to convert urate to urea and glyoxylate, only in the peroxisomes (Noguchi, T., Takada, Y., & Fujiwara, S. (1979) J. Biol. Chem. 254, 5272-5275). The subcellular distribution of these two enzymes involved in further metabolism of urea and glyoxylate in marine animal species was examined by centrifugation in a sucrose density gradient.
Urease was located only in the cytosol of crustacean and mollusc liver; no activity was detected with fish liver.
In fish, crustacean and mollusc liver, the conversion of glyoxylate to glycine may be mainly catalyzed by alanine: glyoxylate aminotransferase. Hepatic alanine: glyoxylate aminotransferase was located both in the mitochondrial matrix and in the cytosol in each species studied.
These findings suggest that peroxisomal urea is transported to the cytosol then degraded to NH₃ for the excretion of purine nitrogens, while peroxisomal glyoxylate is transported to the cytosol or mitochondria then converted to glycine for the reutilization of purine carbons.

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Five components of neutral horseradish peroxidase were isolated and purified by means of column chromatography, and designated B1, B2, B3, C1, and C2, respectively. All the components contained 2 atoms of calcium and 16.8-to-21.0% as much carbohydrate as in the enzyme molecule. They were very similar to one another with respect to physicochemical and chemical properties such as molecular weight, molar absorption coefficient, rate constants of the catalytic reaction and dissociation of cyanide compound, but were dissimilar with respect to isoelectric point. Values of the isoelectric points determined from column isoelectric focusing at 20°C were 5.75 (B1), 7.15 (B2), 7.10 (B3), 9.40 (Cl), and 9.63 (C2). However, these values varied significantly depending upon the method and conditions of the focusing. The acid-alkaline titration curves of components B2 and Cl were flat in the pH region of 6 to 9. The facts suggest that a slight difference in the number of ionized groups of the components causes a large difference in the isoelectric points.

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The dimerization of α-chymotrypsin (α-CT) has been known to involve a specific interaction between the Tyr-146 a-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N^δ1, and the Tyr-146 α-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N^δ1 has a pKa value of around 5.5 and the site between His-57 N^δ2 and the Tyr-146 α-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N^δ1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric α-CT-catalyzed reactions.

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Rat liver microsomal fraction generates ¹⁴CO₂ from [1-¹⁴C] glucose 6-phosphate in the presence of NADP⁺ and a detergent. The activity is mediated through an enzyme system consisting of hexose-6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase inherent to the microsomes, with the latter enzyme reaction being a rate-determining step. Both enzymes of the system in microsomes are extremely resistant to trypsin digestion, thereby distinguishing them from the corresponding cytosol enzymes. A stoichiometric relationship was obtained between the generations of NADPH and ¹⁴CO₂. (2:1 on a molar basis), indicating that the observed generation of NADPH in microsomes could entirely be accounted for by the action of the enzyme system.
A method was devised to measure NADP (H) inside or outside the microsomal vesicles, and it was found that a considerable amount of the cofactor was present within the vesicles. Subfractionation of various intracellular fractions on sucrose density gradients confirmed the close association of NADP (H) with liver microsomes. It is suggested that both enzymes of the system function to generate the reduced form of NADP⁺ in the luminal space of the endoplasmic reticulum, where NADP (H) and glucose 6-phosphate are available.

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Prorenin was purified extensively from porcine kidney by a procedure involving extraction at pH 6.5 in the presence of protease inhibitors, ammonium sulfate fractionation, affinity chromatography on pepstatin-aminohexyl-Sepharose, acetone frac-tionation, chromatography on DEAE-cellulose, gel filtration on Ultrogel AcA 44, chromatography on concanavalin A-Sepharose, and chromatography on CM-Se-pharose. The resulting preparation was essentially homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis. The molecular weight of the preparation was estimated to be 39, 000 by SDS-gel electrophoresis and 44, 000 by gel filtration on Ultrogel AcA 44, and the isoelectric point was at pH 5.1. The purified prorenin was stable for 60 min at 37°C between pH 3.0 and 9.0 and on storage for 4 weeks at-80°C. The prorenin did not show any renin activity which appeared after trypsinization. The molecular weight of trypsin-activated prorenin was estimated to be 40, 000 by gel filtration on Ultrogel AcA 44. The specific renin activity was 2.06 mg angiotensin I/mg of prorenin per h, and the optimal pH for the renin activity was in the pH region from 6.0 to 6.5 with hog and rat plasma angiotensinogens as the substrates. Two distinct isoelectric points were identified at pH 4.9 and pH 5.0 with the trypsin-activated prorenin.

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The insulin binding to hepatic plasma membranes, erythrocytes and adipocytes of 1-, 2-, and 9-month-old rats was measured by using [¹²⁵I] insulin. When rats were fed a fat-free diet, the capacities of the insulin bindings significantly decreased with aging, whereas the dissociation constants were not altered. The capacities of 9-month-old rats were decreased to about half those of 1-month-old rats for plasma membranes, and about one-third for erythrocytes and adipocytes. On the other hand, the plasma insulin levels tended to be high with increasing age at 9-10 a.m., while the levels were even more elevated at 8-9 p.m. and not altered by aging. The insulin binding capacities were increased by feeding the fat-free diet to rats adapted to a stock diet and the increases were reduced with aging. The insulin binding capacities correlated well with the magnitudes of induction of lipogenic enzymes as reported previously [Iritani, N., Fukuda, H., & Fukuda, E. (1981) Biochim. Biophys. Acta 665, 636]. Therefore, it is suggested that insulin binding is implicated in agedependent alterations of lipogenic enzyme induction.

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The composition, topological distribution and biological significance of phospholipids in the membrane of Newcastle disease virus (NDV) grown in embryonated chicken eggs were investigated. Phosphatidylethanolamine and sphingomyelin were the predominant phospholipids in NDV membrane. The location of phospholipids in the lipid bilayer of the membrane was studied by assessing their reactivities with highly purified phospholipase A₂ (Agkistrodon halys blomhoffii) and phospholipase D (Streptomyces chromofuscus), and the biological role of membrane phospholipids was also investigated by using pure phospholipases A₂, C (Bacillus cereus) and D.
Choline-containing phospholipids were found predominantly in the outer layer of the membrane. The inner layer was composed mainly of aminoglycerophos pholipids, though a fair amount of them also appeared to be located in the outer half of the bilayer. When intact virion was treated with phospholipase C, marked decreases in hemolytic activity and infectivity mediated by viral fusion (F) glycoprotein were observed, but hemagglutinating and neuraminidase activities did not change significantly. Apparently complete hydrolysis of phospholipids in the outer half of the lipid bilayer with phospholipase D caused about 22% decrease in the original hemolytic activity. On the other hand, when all phosphatidylcholine and aminoglycerophospholipids in the outer half of the viral membrane were hydrolyzed with purified phospholipase A₂, no significant change in viral hemolytic activity or morphology was detected. No marked change of hemagglutinating and neurami nidase activities was detected on treatment of NDV with phospholipases A₂ and D.
The above results suggest that the integrity of fatty acid ester of glycerophos-pholipids in NDV membrane is not essential for the manifestation of viral activities, though polar groups of the phospholipids in the outer half of the membrane may be involved in the function of fusion (F) glycoprotein, but not in that of hemagglutinat-ing and neuraminidase (HN) glycoprotein of NDV.

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An antibody against blasticidin S (BLS), an antibiotic effective for blast disease of rice, has been produced in rabbits immunized with a blasticidin S-protein conjugate prepared by a novel and convenient procedure devised to couple BLS to bovine serum albumin (BSA) after sodium borohydride reduction of its disulfide bonds, using N-(m-maleimidobenzoyloxy) succinimide (MBS) as a cross-linker. BLS-MBS-BSA conjugate contained about 16 BLS per BSA molecule. Enzyme labeling of BLS with β-D-galactosidase was performed by utilizing another cross-linker, N-(γ-maleimidobutyryloxy) succinimide by means of a convenient labeling method which we introduced last year. A double antibody enzyme immunoassay of BLS which could determine as little as 100 pg per tube of BLS was developed using labeled BLS and anti-BLS antiserum. Various commonly used drugs were found to have little reactivity in this immunoassay, indicating that the anti-BLS produced is highly specific. The titer of the anti-BLS was excellent and 10, 000, 000-fold diluted solution could bind with the enzyme labeled BLS.

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High resolution ¹H NMR spectroscopy at 360 and 400 MHz was used to study the dynamic states of the three methionyl residues (Met 70, 73, and 103) of Streptomyces subtilisin inhibitor (SSI) in a neutral aqueous (²H₂O) solution. Chemical modification and nuclear Overhauser effect studies show that one of the methyl groups exists in the interior of the protein and is surrounded by phenylalanyl residues. With reference to the crystal structure, this methyl group is assignable to that of Met 103 in the hydrophobic core. The other two methionyl residues (Met 70 and 73) are equally susceptible to chemical modifications, but their methyl signals are affected in a different manner by the α-chymotrypsin-cleavage of the peptide bond between Met 73 and Val 74 at the reactive site. The observed linewidths of the methyl groups are consistent with the assumption that the whole side-chain of both Met 70 and 73 are already undergoing rapid internal motions at 20°C, whereas for Met 103 only the rotation of the methyl group is allowed, even above _??_40°C. The highfield shift of the methyl proton resonance of Met 103 by about 0.3 ppm is attribut-able to the ring current effect from the surrounding phenylalanyl residues. This shift is invariant in the temperature range of 40-90°C, showing that the native environment of the methyl group of Met 103 persists up to 90°C at neutral pH. Destruction of the native methionyl environments occurs only above 90°C, and the rate of conversion between the native and the denatured conformations is slower than 10 s^-1. When SSI is mixed with subtilisin BPN' in a one-to-one molar ratio, at least one of the methyl signals of Met 70 and 73 is completely broadened, showing that the side-chain of this methionine is directly involved in the interaction with the enzyme.

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Effects of polydeoxynucleotides on the activity of DNA polymerase-α from sea urchin embryos were studied. Poly (dG), poly (dC) and poly (dC)-oligo (dG) _12-18 inhibited DNA polymerase-a activity in the activated DNA-directed reaction but poly (dA), oligo (dT) _12-18, and poly (dA)-oligo (dT)_12-18 did not inhibit the activity. The inhibitory mode of poly (dC)-oligo (dG)_12-18 or poly (dC) was competitive with activated DNA and that of poly (dG) was noncompetitive with activated DNA. Using poly (dA)-oligo (dT)_12-18 as a template-primer, the inhibition with either poly(dG) or poly (dC)-oligo (dG)_12-18 was competitive with the template-primer. These kinetic results indicate that each of the template-primers tested binds to an identical site on DNA polymerase-α. Similar results were obtained with DNA polymerase-α from HeLa cells.

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The major translation product of rat gastric mucosa RNA in a wheat germ cell-free system was identified as prepepsinogen by electrophoretic analysis of its immunoprecipitate on sodium dodecyl sulfate (SDS)-polyacrylamide gels and amino-terminal sequence determination. The translation product containing radioactive amino acids, purified by SDS-polyacrylamide gel electrophoresis, was shown to have an amino-terminal extension peptide comprising 16 amino acid residues. A partial amino acid sequence of this extension peptide is as follows: Met-X-X-Met-Val-Val-X-Leu-Leu-X-Leu-X-Leu-Leu-X-X-pepsinogen.

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