The hematosides (sialyl-lactosylceramides) of rat small intestine were separated as their acetylated derivatives. The isolated fractions were characterized by mass spectrometry and degradative methods, and the two major fractions also by NMR spectroscopy. From these results hematosides with different sialic acid and ceramide type could be assigned to thin-layer chromatographic bands. This allowed a structural interpretation of the chromatographic patterns observed for different parts of the small intestine. Thus, epithelial cells of ileum contained only hematoside with N-glycoloylneuraminic acid. Duodenum lacked this compound and instead the epithelial cells contained hematoside with N-acetylneuraminic acid. In non-epithelial tissue of both duodenum and jejunum-ileum the major hematoside had N-acetylneuraminic acid. The hematosides of epithelial cells had ceramide containing 18:0 trihydroxy base combined with 16, 20, 22, 24:0, and 24:1 hydroxy fatty acids (major part) or non-hydroxy fatty acids. In the non-epithelial hematosides the ceramide consisted of 18:1 dihydroxy base combined with 16, 18, 20, 22, 24:0, and 24:1 non-hydroxy fatty acids.

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The two phosphoenzymes (E1P and E2P) of Na⁺, K⁺-ATPase were measured as ADP-sensitive and K⁺-sensitive fractions. The sum of these fractions was nearly 1 in the range of 50 to 1, 200mm NaCI. The effects of Na⁺ on the levels of ElP and E2P, on the rate constant of E2P→E1P transition (k₂), on the rate constant of E2P dephosphorylation (k₃), on the rate constant of E1P→E2P transition (k₁) and on the apparent equilibrium constant between E1P and E2P (K_app) were examined. k₁ was found to decrease with increasing Na⁺ concentration, whereas k₂ increased. K_app was found to be directly proportional to the third power of Na⁺ concentration. k₃ increased with increasing Na⁺ concentration and saturated at about 1 M NaCI.
These results are consistent with a simple model in which ATP hydrolysis occurs through effectively only two phosphoenzyme intermediates in the absence of K⁺ and three sodium ions are discharged cooperatively from the enzyme during the E1P→E2P conversion.

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The stimulatory effects of glucose and lactate on protein synthesis by round spermatids (steps 1-8) were further studied. When the cells were incubated with lactate, the response of protein synthesis in round spermatids was closely related to the intracellular level of ATP. The ATP level in spermatids increased to 3.13±0.20 nmol/10⁶ cells from 0.37±0.02 nmol/10⁶ cells after incubation of the cells at 34°C for 60 min in the presence of lactate (20 mM). However, the ATP level fell rapidly to an undetectable level (<0.02 nmol/10⁶ cells) during incubation for 30 min at 34°C without lactate. The ATP level and the rate of protein synthesis in spermatids increased rapidly when lactate (20 mM) was added to the control cells during incubation. It was also found that incorporation of ³²P into ATP was increased by treatment with lactate (20 mM), but glucose (10 mM) had no effect on ³²P incorporation into ATP. When the rates of utilization of glucose and lactate by spermatids were examined, using ¹⁴C-labeled glucose and lactate, the rate of utilization of lactate was faster than that of glucose.
These results suggest that the ATP is probably a major factor in the stimulation of protein synthesis in round spermatids.

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1. Two potent inhibitors of various NADH dehydrogenases including lactate dehydrogenase (I-1 and I-2) were isolated from NADH solution stored at alkaline pH. I-1, which had almost the same affinity for DEAE-cellulose as NADH, was purified after NADH present in the stored solution had been oxidized by acetaldehyde in the presence of immobilized alcohol dehydrogenase, which could not oxidize the inhibitor. 2. I-1 was concluded to be 1, 6-dihydronicotinamide adenine dinucleotide, whereas I-2 was identified as the dimer linked between C-6 of the nicotinamide ring (NC-6) of NADH and NC-2 of I-1. 3. The absorption spectrum of I-1 showed peaks at 345 nm and 260 nm, the ratio of A_345nm to A_260nm being 0.23. The inhibitor was oxidized to equimolar NAD⁺ by phenazine methosulfate (PMS), but not by the NADH dehydrogenases tested. 4. The absorption spectrum of I-2 showed peaks at 338 nm and 260 nm, the ratio of A_338nm to A_260nm being 0.20. It was oxidized only partially by glutamate dehydrogenase or alcohol dehydrogenase but totally by PMS; the oxidations were accompanied by partial and total decreases of the 338-nm peak, respectively, indicating that the inhibitor had two different kinds of oxidation-reduction sites. The extent of inhibition of lactate dehydrogenase by I-2 was not influenced by such oxidation. 5. Of the several NADH dehydrogenases tested, I-1 inhibited all those of the A type and sorbitol dehydrogenase (type, unknown), but not those of the B type, whereas I-2 inhibited lactate dehydrogenase (A type) and malate dehydrogenase (A type), but not alcohol dehydrogenase (A type), glycerol-3-phosphate dehydrogenase (B type), glyceraldehyde-3-phosphate dehydrogenase (B type), glutamate dehydrogenase (B type) or sorbitol dehydrogenase. Of the isoenzymes of lactate dehydrogenase, 4H was more significantly inhibited by I-1 than 4M, whereas both isoenzymes were similarly by I-2.

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A fluorescent ATP analog, 2'-(5-dimethylaminonaphthalene-1-sulfonyl)amino-2'-deoxy ATP (DNS-ATP), was synthesized. In water, the wavelengths of maximum excitation were 260 and 340 nm, and that of maximum emission was 554nm. The fluorescence quantum yield with excitation at 340nm was 0.052. In 80% dioxane-20% water solution, the wavelength of the maximum emission shifted to 527nm and the quantum yield was about 5.4 times in water.
When DNS-ATP was mixed with HMM in the presence of Mg²⁺ ions, the fluorescence intensity of DNS-ATP was enhanced by about 30%, and the wavelength of maximum emission shifted to 545nm. The observed second-order rate constant for the change in fluorescence intensity after adding DNS-ATP to HMM was 1.6×10^-7 M^-1•s^-1, while the observed first-order rate constant for its recovery was 0.17 s^-1. When the HMM DNS-ATPase reaction was measured in terms of the TCA-P₁ liberation, 1 mol of initial burst of P₁ liberation per mol of myosin was observed. In 50mM KCl and at 20°C, the rate of the HMM DNS-ATPase reaction was increased by F-actin from 0.4 to 1.15 s^-1 (in 3mg/ml F-actin). The observed dissociation constant for the binding of DNS-ATP with HMM increased from 1.2 to 20 μM in the presence of 5mg/ml F-actin. However, the extent of change in fluorescence intensity at infinite concentration of DNS-ATP was unaffected by the presence of F-actin.

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The reaction mechanism of beef heart mitochondrial F₁-ATPase was studied by using a fluorescent ATP analog, 2'-(5-dimethylaminonaphthalene-l-sulfonyl) amino-2'-deoxyATP (DNS-ATP), as a substrate. The following results were obtained.
1. In the presence of Mg²⁺, 2 mol of DNS-ATP binds to 1 mol of F₁ with an apparent dissociation constant of 0.44 μm. Upon binding, the fluorescence emission spectrum of DNS-ATP shifted to shorter wavelengths, and the fluorescence intensity at 520nm increased 4.8-fold. On the other hand, in the absence of Mg²⁺, the apparent dissociation constant of F₁-DNS-ATP was 16 μm, and the extent of fluorescence enhancement was 1.3- to 1.9-fold.
2. The initial rate of fluorescence enhancement, v_f, upon addition of Mg²⁺ to a mixture of F₁ and DNS-ATP was given by the following equation:
v_f=^V_f/_{(1+^Kf/[DNS-ATP])}
where V_f=0.34 s^-1 and K_f=3.3 μM. Following the fluorescence enhancement, TCA-P₁ and free P₁ were liberated consecutively. The fluorescence intensity maintained its enhanced level even after the free P₁ liberation and, on addition of an excess amount of EDTA, decreased slowly to the original level. From these findings, the following reaction mechanism for F₁-DNS-ATPase was proposed:
_??_
where an asterisk indicates the state of bound DNS-nucleotide with enhanced fluorescence and E^DNS•ADP*_P is a TCA-unstable reaction intermediate. This reaction mechanism resembles that of myosin ATPase. In accordance with the reaction mechanism and the stoichiometry of DNS-ATP binding, an initial burst of TCA-P₁ liberation of 2 mol/mol F₁, was observed.
3. The addition of an excess of ATP over F₁-DNS-nucleotide in the presence of Mg²⁺ markedly accelerated the liberation of both TCA-P₁ and free P₁. ATP also accelerated the fluorescence decrease of F₁ DNS nucleotide. Direct measurements of released free DNS-nucleotide indicated that the fluorescence decrease was due to the release of DNS-ADP from F₁. ADP, AMPPNP, ITP, and GTP accelerated the F₁-DNS-ATPase reaction in a similar manner. On addition of these NTPs, the release of DNS-ADP from F₁ proceeded in rapid and slow steps. CTP and PPi largely or completely failed to accelerate the conversion of E^DNS•ATP* into E^DNS•ADP*_P Furthermore, CTP and PP₁ induced only the slow release of DNS-ADP. Therefore, we concluded that several nucleotides including ATP bind to the regulatory site(s) on F₁ to induce a conformational change at the catalytic sites and accelerate each of the following steps:_??_On the other hand, PP₁ and CTP mainly accelerate the release of DNS-ADP from E^DNS•ADP* by displacement. AMP did not affect the F₁-DNS-ATPase reaction.
4. The steady-state rate of F₁-DNS-ATPase, v₀, increased linearly with increase in the concentration of DNS-ATP and was 1 s^-1 at 200 μM DNS-ATP, exceeding V_f (0.34 s^-1). The time course of ATP hydrolysis after addition of ATP to F₁-DNS-nucleotide showed no lag phase which corresponds to the release of DNS-ADP. These two findings indicate that F₁-NTPase can also occur through a reaction path in which E^NDP_P is not formed.
5. P₁ enhanced the fluorescence of F₁-DNS-nucleotide and inhibited the displacement of bound DNS-ADP with ATP or PP₁

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Since it has been uncertain whether residue 103 in hen egg-white lysozyme is aspartic acid or asparagine, we reexamined the identity of this residue. To avoid complication, the tryptic peptide T-13 (Ile 98-Arg 112) was further cleaved. The peptide containing residues Gly 102-Arg 112 was obtained by tryptic digestion of lysozyme modified at Asp 101 with diethylenetriamine. The peptide containing residues Ile 98-homoserine 105 was obtained by BrCN treatment of peptide T-13. Both Edman degradation of the former peptide and carboxypeptidase Y digestion of the latter peptide identified residue 103 in hen egg-white lysozyme as asparagine.

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Erythrocyte membrane peripheral and integral proteins have been isolated and purified, and the lateral diffusion of these proteins in a well-defined phospholipid bilayer matrix (dimyristoylphosphatidylcholine) has been studied by fluorescence photobleaching recovery measurements. Our own instrument for the recovery measurements is described and some data for lipid diffusions are compared with those previously reported by other investigators. The peripheral proteins (spectrin and band 4.1) diffuse rapidly on the lipid membrane in its fluid phase. The diffusion constant of _??_5×10^-8 cm²•s^-1 (30°C) was only a little smaller than that for lipid diffusion. The diffusion was greatly slowed down when the host lipid matrix became solid. The integral protein band 3 also diffuses rapidly in the fluid membrane. The diffusion constant of 1.6×10^-8 cm²•s^-1 (30°C) was smaller than those for lipids and for the peripheral proteins. The lateral motion is compatible with diffusion of a cylinder with radius 3 nm in a two dimensional matrix with an inner viscosity of 2 poises and an inner thickness of 4 nm. The band 3 lateral motion was restricted by binding of the cytoskeletal component proteins (ankyrin, spectrin, actin, and band 4.1) to the reconstituted membranes. The diffusion constant decreased to half. The results provide a basis for the elucidation of transmembrane control mechanisms in more complex cellular systems.

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Human κ-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The κ-casein was calcium-insensitive and did stabilize human β-casein and bovine α_s1-casein against precipitation by calcium ions. Formation of micelles from human β-and κ-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-κ-caseins by chymosin was confirmed by SDS-PAGE. Two para-κ-caseins with apparent molecular weights of 13, 000 and 11, 000 appeared. The molecular weight of intact human κ-casein was estimated to be approximately 33, 000. The human κ-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine κ-casein except for serine, glutamic acid, and lysine contents.

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ColEl derivatives carrying cohesive end sites of λ phage genome (=cosλ) can be packaged within λ phage particles. The DNA structure of the prototype ColEl-cosλ derivative named pKY2257 was studied because of its potential usefulness in various fields in molecular biology.
pKY2257, which carries an intact galactose operon of E. coli, is a convenient replicon to detect Tn3 translocation. It was found that one of the pKY2257::Tn3 derivatives, pKY2113, generated various small plasmids in E. coll. The molecular structures of some of these deletion mutants were compared with each other and with those of parental plasmid DNAs by heteroduplex analysis and restriction enzyme digestion. A possible mechanism, which seems to be unique to this kind of deletions, is discussed on the basis of the present results.

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Human prothrombin contains three asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB³H₄ reduction. All of the oligosaccharides contain N-acetylneuraminic acid. The neutral oligosaccharides obtained from all acidic oligosaccharides by sialidase digestion are identical. By the combination of sequential exoglycosidase digestion and methylation analysis, the structures of the asparagine-linked sugar chains of human prothrombin were confirmed to be as follows:
NeuAcα 2→6 Galβ1→4GlcNAcβ1→2 Manα l→6 (NeuAcα2→6 GaIβ1→4GlcNAcβ1→2Manα1→3) Manβ1→4GlcNAcβl→4GIcNAc, NeuAcα2→6Galβ1→4GIcNAcβ1→2Manα1α6(Galβ1→4G1cNAcβl→2Manαl→3)Manβ1→4GlcNAcβ1→4GIcNAc, and Galβ1→4GIcNAcβ1→2Manα1→6(NeuAcα2→6Gal→1→4GlcNAcβ1→2Mana l→3)Manβ1→4GIcNAcβ1→ 4GlcNAc.

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Pure recA protein was easily obtained from Escherichia coli harboring plasmid pTM-2 which carried the recA gene by two chromatographic steps on phosphocellulose and DEAE-cellulose. RecA protein was stable in the pH range of 6 to 9 at 25°C. RecA protein was found to aggregate highly under these conditions. Lowering of the protein concentration, the presence of glycerol, and lowering of the pH in the pH stability region diminished the extent of aggregation.
The spectroscopic properties of recA protein were measured in the presence of 10% (v/v) glycerol. RecA protein had an absorption maximum at 278nm. The value of A^1%_1cm at 278nm was determined to be 5.7. The tryptophyl fluorescence spectrum excited at 295nm had an emission maximum at 340nm and the quantum efficiency of recA protein relative to N-acetyl-L-tryptophanamide was determined to be 0.65. The CD spectrum of recA protein had negative double maxima at 210 and 220nm. The α-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250nm.
All three cysteinyl residues of recA protein were reacted with 5, 5'-dithiobis (2-nitrobenzoic acid), and recA protein was found to have neither intramolecular nor intermolecular disulfide bond. The reactivities of the SH groups were changed by the presence of ATP or ADP.
The denaturation of recA protein by guanidine hydrochloride was studied by measuring CD at 220nm and tryptophyl fluorescence. The denaturation curve obtained by CD measurement consisted of two stages, one of which lies between 0 and 1.8M and the other above 1.8M guanidine hydrochloride. On the other hand, the denaturation curve obtained by fluorescence measurement consisted of a single transition in the concentration range of about 1 to 2.3M guanidine hydrochloride. RecA protein plays a variety of important roles in the biological function of Escherichia coli (1, 2). Recently the nucleotide sequence of the recA gene was determined by Horii et al. (3) and hence the amino acid sequence. RecA protein consists of 352 amino acid residues and the molecular weight is calculated to be 37, 842. Purified recA protein shows a variety of ATP-dependent activities in vitro: ATPase activity dependent upon singlestranded DNA (1, 4); pairing activity between homologous DNA molecules (5-8); and protease activity for phage repressors (9, 10) and lexA protein (a repressor for SOS functions) (11, 12) in the presence of single-stranded DNA. To correlate the biological functions with the structure of recA protein, we began with the physicochemical characterization. RecA protein has been prepared by Roberts et al. (4), Weinstock et al. (5), Shibata et al. (6), and Ogawa et al. (13). However, the quantity of the enzyme isolated by these methods has not been sufficient to permit characterization. In the present study we succeeded in preparing the pure enzyme in large quantity and quite easily.
The stability of recA protein at various pH values was also studied by measuring its ATPase activity. It was found that recA protein is stable in the pH region of 6 to 9 at 25°C. Ogawa et al. (1) found that the aggregation state of recA protein affects the ATPase activity. We studied the aggregation state of recA protein by Sephacryl S-300 gel chromatography and found that recA protein aggregates highly even at 1×10^-6M in the presence of 10% (v/v) glycerol and in the pH region of 6 to 9. Under these conditions, the ultraviolet absorption, fluorescence, and circular dichroism were measured. RecA protein has three cysteinyl residues. Although the importance of the SH group(s) in the catalysis has been suggested by Weinstock

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The effect of various concentrations of heroin on globin synthesis was studied at various concentrations of potassium salts using a rabbit reticulocyte lysate system. The optimal concentration of hemin for globin synthesis was 10-30 μM at 100mM and 200mM K⁺.
When the concentration of potassium salts was changed, globin synthesis without added hemin was optimal at 200mM K⁺ or at 160mM K⁺ and 60mM Cl-and the globin synthesis at higher concentrations of potassium salts was similar to that at the optimal concentration of hemin.
Using rabbit reticulocyte lysate, the binding of [³⁵S]Met-tRNAf to the 40 S ribosomal subunits was compared with that at the optimal concentration of added hemin. It was found that the binding without added hemin was inhibited greater at 100mM KOAc than at 200mM KOAc. But, a high concentration of hemin inhibited the binding at both KOAc concentrations.
The postribosomal supernatant of the lysate was preincubated at various concentrations of KOAc and the inhibitory activity of the supernatant was measured in the lysate globin synthesis system. The data suggested that so-called “hemincontrolled translational inhibitor” was slowly formed and less active at the high KOAc concentration. Therefore, it is also suggested that stimulation of globin synthesis and Met-tRNA_f binding to 40 S ribosomal subunits in hemin-deficient lysate are due to the slower formation of HCI and the lower activity of HCI at a high K⁺ concentration.
On the basis of these observations, 1) the different requirements of heroin for globin synthesis between reticulocytes and their lysates and 2) a possible physiological meaning of the findings are discussed.

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1. From a digestive produced from Aspergillus saitoi, a minor glucoamylase [EC 3. 2. 1. 3] named Gluc M₂ was purified in a yield of 6%, besides a major glucoamylase named Gluc M₁ (M. W. 90, 000) which was previously isolated in a yield of 21%. The purified enzyme was proved homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, ultracentrifugation, immunodiffusion and also from the absence of the glycosidase activities detected in the crude extract. 2. The pH optimum of Gluc M₂ was 4.5 with soluble starch as a substrate. The enzyme was stable between pH 2.5 and 7.5 and retained full activity at temperatures up to 50°C. Gluc M₂ was denatured almost completely with 5 M guanidine hydrochloride but only partially denatured with 8M urea. 3. Glue M₂ was a glycoprotein containing 0.55% glucosamine and 12%. neutral sugar which consisted of a large amount of mannose and small amounts of galactose and glucose. The molecular weight of the enzyme was estimated to be about 70, 000 by SDS-polyacrylamide gel electrophoresis and its amino acid and sugar compositions. 4. Although differing in both amino acid and sugar compositions and also in molecular weight, Gluc M₂ and Gluc M₁ had almost identical isoelectric points and showed a common antigenicity in immunodiffusion. The N-terminal amino acid sequences of both enzymes were the same: H₂N•Ala-Val-Ile-Val-. 5. The kinetic parameters, K_m and V_max, of Gluc M₂ for the low-molecular-weight substrates were similar to those of Gluc M₁. In contrast, both K_m and V_max values of Gluc M₂ for the high-molecular-weight substrates were consistently larger than those of Gluc M₁, the difference was most pronounced when glycogen was the substrate and less pronounced when amylopectin was the substrate.

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Cytochrome P-450 was solubilized from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B and heparin-Sepharose CL-6B columns. Fatty acid ω-and (ω-1)-hydroxylation activity was reconstituted from the partially purified cytochrome P-450 and NADPH-cytochrome c reductase, with phosphatidylethanolamine or phosphatidylcholine. The activity was further stimulated by addition of detergent-solubilized cytochrome b₅ from rabbit liver microsomes. Trypsin-solubilized or boiled detergent-solubilized cytochrome b₅ had no effect. Among fatty acids tested, caprate, laurate, myristate, and palmitate were the most effective substrates. When caprate and laurate were used as the substrates, the products were the corresponding ω-and (ω-l)-hydroxy fatty acids. The ratio of these products was altered by addition of cytochrome b₅. On the other hand, when myristate and palmitate were the substrates, small amounts of unknown polar fatty acids were also formed besides ω-and (ω-1)-hydroxy fatty acids, and the ratio of these products was not affected by addition of cytochrome b₅. Benzo(a)pyrene hydroxylation activity was also reconstituted from the same cytochrome P-450 preparation, NADPH-cytochrome c reductase, and phosphatidylserine. However, cytochrome b₅ showed only a slight stimulation. The possibility that different cytochrome P-450 species are involved in fatty acid and benzo(a)pyrene hydroxylations is discussed.

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An NADPH-specific aromatic aldehyde-ketone reductase located in guinea pig liver microsomes can be effectively solubilized with nonionic detergents, but not with bile salts and hydrolytic enzymes. Destruction of microsomal membranes by nonionic detergents or acetone treatment leads to significant activation of the reductase, indicating that the enzyme is partly latent in intact microsomes. After solubilization with Triton X-100, the reductase has been highly purified. The purified enzyme catalyzes the NADPH-linked reduction of xenobiotic aromatic aldehydes and ketones as well as 3-ketosteroids, notably 5α- and 5β-dihydrotestosterones. The reductase activities for xenobiotic carbonyl compounds and for 3-ketosteroids are each inhibited by addition of the other type of substrate and show the same pH optimum, cofactor requirement, and heat stability, indicating the same enzyme is responsible for the reduction of the two types of substrates. Hexose-6-phosphate dehydrogenase, purified from guinea pig liver microsomes, acts as a more effective NADPH generator for the reductase than yeast and guinea pig liver cytosolic glucose-6-phosphate dehydrogenase. Evidence has been obtained that hexose-6-phosphate dehydrogenase undergoes a functional interaction with the reductase, facilitating the provision of NADPH to the reductase activity both in the reconstituted system and in microsomes.

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Gramicidin S (GS) containing ¹⁴C-labeled praline was synthesized by a solid-phase method, and the labeled GS dihydrochloride was obtained as crystals. The labeled GS exhibited same antibacterial activity as natural GS. Strains sensitive to GS (B. subtilis and S. aureus) and an insensitive strain (E. coli) were treated with the labeled GS, and the amount of the labeled GS adsorbed on the cells was measured. GS was adsorbed rapidly on the cells of the sensitive strains; the amount adsorbed increased linearly with GS concentration up to 1-1.5 μg/ml, and at a lower rate at above 1.5 μg/ml. GS molecules covered most of the cell surface at the minimum inhibitory concentration of 1.5 μg/ml ; the number of molecules adsorbed per cell was 1.3-1.4×10⁶. No GS was adsorbed by the insensitive strain.

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An alkali-insoluble core material was isolated from the fruit bodies of Lentinus edodes after exhaustive extraction with 24%. NaOH at 5°C. This material consists mainly of glucan which is closely associated with chitin. Methylation analysis has shown that the glucan part of the core material (skeletal glucan) has a highly branched structure with 1, 6 and 1, 3 linkages in a molar ratio of 2:1. Stepwise enzymatic hydrolysis with basidiomycete sp. QM 806 β-1, 3-glucanase has indicated the heterogeneity of the skeletal glucan. The outer part of the skeletal glucan seems to be composed mainly of β-1, 3 and β-1, 6 glucoside linkages and has a close structural similarity to lentinan, a water-soluble β-glucan from L. edodes. The middle part of the skeletal glucan appears to be composed mainly of β-1, 6 glucosidic linkages. The innermost part of the skeletal glucan is a highly branched glucan with β-1, 6 and β-1, 3 linkages. Probably, it is associated with chitin and a small amount of amino acid polymer.

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Amino acid and potassium transport during progressive starvation has been investigated in monolayer cultures of hepatocytes with either αα-[1-¹⁴C]aminoisobutyric acid (AIB) or ⁸⁶rubidium used as a tracer for K⁺. In the presence of sodium (Na⁺-dependent “A”-system), the rates of amino acid uptake were gradually increased with progression of starvation. When Na⁺ was replaced by choline (Na⁺-independent component), the transport was diminished, and accumulation was not affected by fasting. Computer analyses of the kinetic data revealed the emergence of a high-affinity, low-K_m component for the AIB transport during early stages of fasting (24 h: 0.59mm; 48 h: 0.97mm), but the low-K_m system disappeared after extended periods of starvation (72 h). Low-K_m and low-affinity, high-K_m parameters are affected by either culture or nutritional conditions, e.g. culture media containing different concentrations of amino acids, and changes in the contribution of the two individual components to the total rates of the Na⁺-dependent AIB transport are reported. Rates of Rb⁺ transport were concomitantly increased during progressive starvation, and were affected in vitro by culture media containing high or low levels of amino acids. The enhanced and ouabain-sensitive Rb⁺ transport reflects an increased activity of the electrogenic Na-K-pump during progressive starvation. Kinetic analyses of these data indicate multiple and cooperative binding sites of the enzyme (Na⁺, K⁺-ATPase) involved in the K⁺ uptake, and in addition, since the Hill coefficient n is>1.0, the transport system is positively cooperative. Enhanced rates of Rb⁺ transport during starvation also reflect changes in the electrochemical gradient for Na⁺ and, with this, they suggest alterations in the membrane potential across hepatocellular membrane; the latter is most likely the apparent driving force for the increased AIB transport in the Na⁺-dependent “A”-system. The low-K_m component with its considerable contribution to the amino acid transport in the physiological range provides the liver with a highly potent emergency system during progressive starvation.

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When EF-Tu was photooxidized for 20 min at 0°C in the presence of 10 μm GDP and 5 μM rose bengal, the activity to promote the binding of [¹⁴C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [³H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [¹⁴C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site (s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA•EF-Tu•GTP complex.
Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu.GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.

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The pH dependence of the binding constant of Ca²⁺ to the phospholipase A₂ of A. halys blomhoffii was studied at 25°C and an ionic strength of 0.1 by the tryptophyl fluorescence method and aromatic circular dichroism (Ikeda and Samejima (1981) J. Biochem. 89, 1175-1184), and was compared with those for cobra venom phospholipases A₂ (Teshima et al. (1981) J. Biochem. 89, 13-20) and for porcine pancreatic enzyme (Pieterson et al. (1974) Biochemistry 13, 1439-1445). The shape of the pH-dependence curve was closer to that for the porcine enzyme than those for the cobra enzymes. The data were analyzed on the basis of our previous findings (Ikeda and Samejima (1981) J. Biochem., 90, 799-804) that the pK value of an ionizable group (α-amino group) is perturbed from 7.30 to 6.30 on the Ca²⁺ binding, and that the protonation of another group corresponding to Asp 49 of the porcine enzyme with a pK value of 5.16 competes with the Ca²⁺ binding. An additional ionizable group with a pK value of 6.45 was found to participate in the Ca²⁺ ion binding, and this was assigned to the His residue corresponding to His 48 in the active site of the porcine enzyme.

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Recently we found that diadenosine 5', 5'''-pl, p4-tetraphosphate inhibits ADP-ribo-sylation of histone Hl by poly (ADP-ribose) polymerase and that the apparent inhibition was due to a preferential ADP-ribosylation of this nucleotide in place of histone Hl under the reaction conditions used (Yoshihara, K. and Tanaka, Y. (1981) J. Biol. Chem. 256, 6756-6761): although ADP-ribosylation of histone Hl itself was limited, the presence of histone in the reaction mixture was essential for this nucleotide to be modified. In this respect, all five classes of calf thymus histories and a basic homopolyamino acid, poly-L-lysine, were more or less effective, although bovine serum albumin was not. A chase experiment indicated that ADP-ribose covalently bound to histone initially in the reaction could not be transferred to a nucleotide during a chase. Thus, histone does not seem to function as an intermediate carrier for ADP-ribose to be transferred to a nucleotide. Since an equilibrium dialysis of histone Hl against a buffer containing ³H-labeled diadenosine 5', 5'''-pl, p4-tetraphos-phate revealed that this nucleotide could be bound to histone Hl, binding of the nucleotide to a protein may be essential for the nucleotide to be ADP-ribosylated by the enzyme.
Using this reaction system, we also examined the ADP-ribosylation of other nucleotides with similar structure to the diadenosine nucleotide: in addition to this compound, its analogs (diadenosine oligophosphates), diguanosine 5', 5'''-pl, p4-tetraphosphate, and ADP-ribose were found to be effectively ADP-ribosylated in the enzyme reaction. Though a very low extent of ADP-ribosylation of 5'-ATP and 5'-GTP was also detected, 5'-UTP, 5'-dATP, 5'-dGTP, and 5'-dTTP were all ineffective..
The present study demonstrated that not only proteins but also some low molecular nucleotides of suitable structure, such as dinucleotide polyphosphates, can serve as good acceptors for ADP-ribose in the presence of histone in the poly (ADP-ribose) polymerase reaction.

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High pressure column chromatography was applied to estimate the bound nucleotides on F₁-ATPase. In this way, various species of adenine nucleotide in 0.1 to 0.3mg of enzyme protein were well separated and estimated with a precision of about 10 pmol within 10 min. ATPase in submitochondrial particles contained 2 mol of ATP and 1 to 2 mol of ADP. The nucleotide content and binding nature of the enzyme varied at different stages of purification, but a total of four binding sites were found in enzyme preparations at all steps. Highly purified enzyme had two tight binding sites for ATP and two loose binding sites, one for ATP and one for ADP. The tight binding of ADP observed in submitochondrial particles was reconstituted by continuous supply of ATP and Mg²⁺ to the purified enzyme. Removal and rebinding of the nucleotides did not affect ATP-hydrolyzing activity but caused conformational changes of the enzyme, as demonstrated by measuring cold-lability and trypsin digestion. An analog of ATP, AMPP (NH) P, was found to bind to loose binding sites of the purified enzyme with 2 mol of tightly bound ATP, and to inhibit ATP-hydrolyzing activity competitively. The analog also bound to the tight sites under special conditions, protecting the enzyme against cold inactivation. During enzymatic hydrolysis of [³H] ATP, labeled ATP and ADP were both bound at the loose sites, but only slight amounts of these nucleotides were bound to the tight sites. From these results it is inferred that the loose sites are catalytic, while the tight sites are not.

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F₁-ATPase inhibitor was purified from yeast, Saccharomyces cerevisiae. The purified inhibitor blocked ATPase activity in the presence of ATP and Mg²⁺ by forming a latent equimolar enzyme-inhibitor complex with ATP and ADP newly bound to loose sites on the enzyme. A small portion of externally added ATP was hydrolyzed before the latent complex was formed but the hydrolysis was not directly related to the complex formation. Newly bound ATP tended to be converted to ADP when the ATP concentration of the medium was low. ATP tightly bound to the enzyme was not directly involved in formation of the complex. The complex was fairly stable in the presence of excess inhibitor and ATP but at a high concentration of the enzyme (10^-5 M), the inhibition was not complete, although only about 0.03% of the original activity remained unblocked.

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The amino acid sequence of an intrinsic inhibitor of mitochondrial ATPase isolated from yeast was completed by using solid-phase sequencing and conventional procedures. The inhibitor was found to be composed of 63 amino acid residues, to lack tryptophan, cysteine, and tyrosine, and to have a molecular weight of about 7, 383. The inhibitor was characterized as a basic protein with 16 basic and 13 acidic amino acid residues, and several clusters of basic residues were noted. Some comments are made on the hydrophobic amino acids and the presence of repeated sequences.

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The rate constants of the cleavage of glycosidic linkage, hydration (hydrolysis) and transglycosylation in a lysozyme-catalyzed reaction of substrate chitooligosaccharides were evaluated by computer analysis of the experimentally obtained reaction timecourses. In the computer analysis, the rate equation was numerically solved by use of the known binding constants for each subsite. Because of the complexity of the lysozyme-catalyzed reaction, optimal values of rate constants were determined by checking the sensitivity of each rate constant to the computed time-courses. It was not possible to estimate uniquely the rate constants of transglycosylation and hydration, owing to the nature of the enzymatic reaction, but it was possible to estimate accurately their ratio. The estimated values were 0.94 s^-1 for the rate constant for the cleavage of glycosidic linkage and 133 for the ratio of rate constants of transglycosylation and hydration.

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In order to clarify the interaction of membrane proteins in intact cells (not ghosts), a new bifunctional cleavable reagent, maleimidomethyl-3-maleimido propionate (MMP), was synthesized. Human intact erythrocytes and ghosts were incubated with this reagent. Cross-linked products were analyzed by two-dimensional polyacrylamide gel electrophoresis with incubation of the first dimensional gel in 0.1 N NaOH for 15 min at room temperature before the second run. In the case of ghosts, newly appeared bands were tentatively identified as a spectrin dimer, a complex of band 2 with band 5°, which runs very slightly more slowly than band 5 and is phosphorylated by ³²P in intact cells, a complex of band 3 with band 5, a dimer of band 3 and a dimer of band 5°. In the case of intact cells, the newly appeared bands were complexes of band 3 with bands 4.3, 4.5, 4.6, and 4.7 and band 5 dimer, in addition to those seen in the case of ghosts. The technique of membrane phosphorylation permitted the clearer identification of each band. This reagent is useful for studying the relationship among cell membrane proteins in intact erythrocytes.

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A macroglycolipid containing galactose and N-acetylglucosamine as predominant sugar constituents was prepared together with glycophorin from rabbit erythrocyte membranes by extraction with lithium diiodosalicylate and partition in aqueous phenol. The macroglycolipid was effectively separated from the glycophorin fraction by ion-exchange chromatography in the presence of a detergent, Ammonyx LO. Its yield (ca. 4mg/100ml erythrocytes) was significantly higher than that of the macroglycolipids from human erythrocytes. The structure of the carbohydrate moiety in the macroglycolipid was analyzed by methylation analysis, Smith degradation, nitrous acid deamination, and chromium trioxide oxidation. Assuming one ceramide residue per molecule, the average number of sugars in the macroglycolipid was about 30. The macroglycolipid had a highly branching structure: Gal (α1-3) Gal(β1→4)GIcNAc sequences are present at nonreducing termini and→3Gal(β1-4) GlcNAc repeating units are present in the inner core of the sugar chain. Some of tnner galactose residues branch at the C-6 position. Constituents of the ceramide moiety were also characterized.

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Insulin induced tyrosine aminotransferase (TAT) activity in cultured rat hepatoma cells (R117-21B). The induced enzyme activity reached a maximum at 6 to 8 h and then decreased to the control level after 24 to 48 h of incubation. The degree of the induction of TAT was related to the insulin concentration in the range of 0.005 to 0.5 ng/ml, and above a concentration of 0.5 ng/ml the enzyme activity reached a plateau at 6 h of incubation. However, the binding of insulin to its receptors increased continuously after the maximum induction was observed. The number of high affinity insulin binding sites in R 117-21 B cells was 2.5×10⁵ per cell, and the apparent dissociation constant (K_d) of the insulin receptor system at 25°C was 3.7 nM.
It was revealed that, when only 1.9% of the total high affinity insulin binding sites were occupied by insulin in R 117-21 B cells, a maximum response in terms of enzyme induction occurred. Under these conditions, 3, 900 insulin molecules were taken up by each cell to yield the maximum induction of TAT.
When insulin alone was readded to the culture after disappearance of the insulin effect, the enzyme activity increased only slightly or did not change. However, if insulin was added in a fresh culture medium, the enzyme activity increased normally. This indicates that a certain component(s) in the conditioned medium inhibited the reinduction of TAT by fresh insulin. However, when insulin-untreated fresh cells were incubated with insulin in the conditioned medium, the induction of TAT was observed. These data show that some cellular modulation occurred during the insulin treatment as well, and that this modulation and the certain component(s) contained in the conditioned medium inhibited the reinduction of TAT activity by fresh insulin.
The magnitude of the inhibitory effect of the conditioned medium on the reinduction of TAT by fresh insulin was related to the ratio of the conditioned medium to fresh medium during the induction of the enzyme. The unknown component(s) contained in the conditioned medium was heat-stable and dialyzable, and was not extracted with ethanol or acetic acid from the lyophilized conditioned medium.
It was suggested, therefore, that insulin induces intracellular modulations in the hepatoma cells, and that the unknown inhibitory component(s) contained in the conditioned medium also contributes to the regulation of TAT by insulin.

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The amino acid sequence of human spleen histone H3 was investigated as a study in histone evolution, following previous investigations of human spleen histone H2B [Ohe, Y., Hayashi, H., & Iwai, K. (1979) J. Biochem. 85, 615-624] and H2A [Hayashi, T., Ohe, Y., Hayashi, H., & Iwai, K. (1980) J. Biochem. 88, 27-34]. The H3 fraction was obtained as described previously and further purified by Bio-Gel P-10 chromatography. The reduced and carboxymethylated H3 was digested with an arginine-specific protease, Clostripain, and the peptides were fractionated by repeated column chromatographies with reasonable recoveries. Most of these peptides, together with two tryptic peptides and one chymotryptic peptide obtained similarly, were sequenced by Edman degradation. Thus, the human H3 was deduced to have a sequence of 135 amino acid residues identical with that of the main component of calf thymus H3, except for one complete substitution (Ser for Cys-96) and four partial substitutions (Ser for Ala-31, Ala for Ser-87, Ile for Val-89, and Gly for Met-90), which suggest the presence of at least two variants. Three methylated lysines (residues 9, 27, and 36) and two acetylated lysines (residues 14 and 23) were also found. The substitutions and modifications found in the human H3 are compared with those in the known H3 sequences of other eukaryotes, and the implications are discussed.

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The amino acid sequence of a photosynthetic cytochrome c-553 isolated from a brown alga, Petalonia fascia was determined by BrCN fragmentation and a solid phase Edman degradation. The cytochrome contains 85 amino acid residues, giving a molecular weight of 9, 803. The complete amino acid sequence is as follows: Val-Asp-Ile-Asn-Asn-Gly-Glu-Ser-Val-Phe-Thr-Ala-Asn-Cys-Ser-Ala-Cys-His-Ala-Gly-Gly-Asn-Asn-Val-Ile-Met-Pro-Glu-Lys-Thr-Leu-Lys-Lys-Asp-Ala-Leu-Glu-Glu-Asn-Glu-Met-Asn-Asn-Ile-Lys-Ser-Ile-Thr-Tyr-Gln-Val-Thr-Asn-Gly-Lys-Asn-Ala-Met-Pro-Ala-Phe-Gly-Gly-Arg-Leu-Ser-Glu-Thr-Asp-Ile-Glu-Asp-Val-Ala-Asn-Phe-Val-Ile-Ser-Gln-Ser-Gln-Lys-Gly-Trp. The highest homology was found between the sequences of cytochromes c-553 of P. fascia and Alaria esculenta, the next between those of P. fascia and Porphyra tenera.

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Two different classes of gizzard heavy meromyosins (HMMs) were prepared from phosphorylated myosin by chymotryptic digestion in the presence and absence of ATP and were compared with respect to their actin-activated Mg²⁺-ATPase reactions. One class of HMM, named HMM(+), had a cleavage at site 1 in the N terminal portion of the heavy chain and the other class of HMM, named HMM(-), had no cleavage at this site. Maximum turnover rate (V_max) of the skeletal actogizzard HMM Mg²⁺-ATPase reaction was obviously different between HMM(+) and HMM(-). The V_max value of HMM(+) was 2.5-fold larger than that of HMM(-). On the other hand, the apparent association constants (K_a) of skeletal muscle actin for both HMMs which were deduced from double reciprocal plots (v^-1 versus [actin]^-1) seemed to be identical. The difference in V_max value was attributed to the cleavage at site 1 since a following chymotryptic cleavage of HMM(-) at site 1 caused a 2.5-fold increase in the V_max value. That site 1 in the N terminal portion of the gizzard myosin heavy chain was the key locus for the actin-myosin interaction was shown in addition to our previous finding of the effects of cleavage at site 1 on the ATPase activity and nucleotide binding ability of gizzard HMM (Okamoto, Y. & Sekine, T. (1981) J. Biochem. 90, 833-842; 843-849).

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The recent communication by Bishop and Gray (J. Biochem. 90, (1981)) on the streaming in the circular slit of stream cells is probably based on observations under conditions where no active streaming can be produced because of a substantial lack of F-actin. According to our experience, these conditions are similar to those obtained when actins are partly denaturated. Hence, for the sake of their criticism, detailed comparisons should be added of the stability of streaming, the distribution of streaming velocities as well as their sizes, acto-HMM ATPase activities under various streaming velocities and the amount of proteins fixed to the Millipore filter.

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Mitochondrial ornithine aminotransferase was synthesized in a rabbit reticulocyte cell-free system using polysomal mRNA from rat liver. Upon analyzing the primary translation product by electrophoresis and fluorography, we found that ornithine aminotransferase is synthesized not as a large precursor but as one with its authentic size. Free ribosomes were found to be the main site of synthesis of ornithine aminotransferase.

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We studied the metabolism of radioactive stearic acid by cultured skin fibroblasts from patients with adrenoleukodystrophy (ALD) and its variant, adrenomyelo-neuropathy (AMN), to clarify the mechanism of the increased content of very long chain saturated fatty acids in cholesterol esters and sphingolipids, which are known to be the characteristic biochemical changes in ALD and AMN. A substantial amount of hexacosanoic acid (C 26:0) was synthesized from stearic acid by ALD and AMN fibroblasts, whereas only a trace amount of hexacosanoic acid was synthesized by control fibroblasts. This indicates that the primary biochemical defect in ALD and AMN may involve the elongation system of very long chain fatty acids.

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