The cytochromes of anaerobically grown yeast cells were studied; cytochrome b₅ and P-450 were recovered in the fraction which was precipitated between 10, 000×g and 105, 000×g and were identified as yeast microsomes by chemical analysis and electron microscopic examination. These cytochromes from the microsomes were reduced by either NADH or NADPH and the reduced cytochromes were readily oxidized by air. Moreover, evidence for the presence of NADH-cytochrome b₅ reductase and NADPH-cytochrome c reductase was obtained.
From these results, it was concluded that microsomes of anaerobically grown yeast cells contain the same electron-transferring components as the microsomes of mammalian hepatocytes, i.e., cytochrome b₅, P-450, NADH-cytochrome b₅ reductase [EC 1. 6. 2. 2] and NADPH-cytochrome c reductase [EC 1. 6. 2. 4]. These components may comprise an electron-transport system resembling that of hepatic microsomes.

View full abstract

A b-type cytochrome previously called cytochrome b was localized in the microsomes from anaerobically grown yeast cells. The cytochrome was solubilized from the microsomes by tryptic digestion, and was highly purified.
The spectral properties of the cytochrome agreed well with those of cytochrome b₅ from liver microsomes.
Molecular weight, midpoint oxidation-reduction potential and net charge of the purified cytochrome were also similar to those of cytochrome b₅ purified from rat liver microsomes by similar procedures.
The purified cytochrome could be reduced by NADH-cytochrome b₅ reductase [EC 1. 6. 2. 2] from mammals.
The results indicated that the cytochrome should be named cytochrome b₅ of yeast.

View full abstract

O-Acetyl-L-homoserine (OAH) sulfhydrylase was highly purified from baker's yeast by a modification of our previous method. When assayed in phosphate buffer, but not in Tris-HCl buffer, the purified enzyme catalyzed the O-acetyl-L-serine (OAS) sulfhydrylase reaction at a rate which was about 15% that of the OAH sulfhydrylase reaction. The apparent absence of OAS sulfhydrylase activity in Tris-HCl buffer was found to be due to the instability of OAS in this buffer at alkaline pH's. Throughout the purification steps the OAH and OAS sulfhydrylase activities were purified in parallel, and neither DEAE-cellulose column chromatography of a partially purified preparation nor Sepharose 4B gel filtration of the final preparation could separate the two activities. Upon polyacrylamide gel electrophoresis the purified enzyme was separated into three protein bands, of which only the main band possessed the two activities. Moreover, the two activities behaved in the same way on heat treatment. Finally, the levels of OAS sulfhydrylase activity in extracts of various methionine auxotrophs of yeast varied in parallel with those of OAH sulfhydrylase activity. It is concluded that OAS and OAH sulfhydrylase activities of yeast are catalyzed by the same enzyme protein.

View full abstract

The chain compositions of invertebrate collagens from the foot muscle of abalone, the skins of octopus and squid, and the subcuticular membranes of swimming crab and spiny lobster, were studied by CM-cellulose chromatography of the denatured collagens and by chemical analysis and disc gel electrophoresis of their chromatographic fractions. These invertebrate collagens were solubilized by limited digestion with pepsin [EC 3. 4. 23. 1] and found to be assembled from three α chains. The abalone collagen showed the chain composition [α]₃, while the other four collagens had the composition [(αl)₂α2] commonly found in various vertebrate collagens. The squid and lobster collagens, however, contained another species of α chain (α1'), which is chromatographically separable from α1 chain. Both chains have a very similar amino acid compositions and glycosylated hydroxylysine contents.
These invertebrate collagens contained more glycosylated hydroxylysines (largely glucosylgalactosylhydroxylysine) than most vertebrate collagens. In the invertebrates, α2 chains were unique in possessing higher contents of hydroxylysine and glycosylated hydroxylysines as compared with αl chains.

View full abstract

Arylsulfatase A [EC 3. 1. 6. 1] was purified from boar testes. The enzyme was extracted with 10m_M Tris-HCl buffer (pH 7.8) and fractionated with ammonium sulfate, acetone and by DEAE-cellulose column chromatography. Arylsulfatase A activity was measured with 4-nitrocatechol sulfate. ³⁵S-Labeled sulfatide and seminolipid were used as natural substrates. Arylsulfatase A activities toward 4-nitrocatechol sulfate and two other natural substrates were contained in the same fraction at every purification stage. It was suggested that seminolipid was a physiological substrate of arylsulfatase A in the testis.

View full abstract

The stoichiometry of the reaction of stripped luciferase from Photobacterium phosphoreum was determined to be luciferase: FMNH₂: O₂=1:1:1. A red shift of FMNH₂ absorption bands and quenching of protein fluorescence were observed on binding of FMNH₂ to luciferase. The inhibitory effects of FMNH₂ derivatives on the luciferase reaction suggested that the isoalloxazine nucleus, ribitol moiety and phosphate group are all involved in the binding of FMNH₂ to luciferase. The dissociation constant of the luciferase-FMNH₂ complex, estimated by various titrimetric measurements, was 1-6×10^-7_M. The affinity of FMN to luciferase was less than 10^-5-fold that of FMNH₂. The standard potential of luciferase-bound FMN/FMNH₂ was calculated to be -0.06V.

View full abstract

Purified formate dehydrogenase was prepared from dry seeds of pea (Pisum sativuni). The enzyme preparation was more than 95% homogeneous in terms of acrylamidegel disc electrophoresis and the molecular weight was found to be 70, 000±2, 000 by gel filtration and 72, 000±2, 000 by sedimentation equilibrium.
The pH-activity curve, spectral properties, and amino acid composition were investigated. The enzyme was colorless and contained no significant amounts of iron, manganese, and zinc. Four moles of PCMB (p-chloromercuribenzoate) reacted with the enzyme and the activity of the enzyme was almost completely inhibited as a result. The reaction with PCMB was retarded in the presence of NAD⁺. On the other hand, DTNB (dithio-bis-nitrobenzoate) was found to react with sulfhydryl groups of the enzyme much faster in the presence of NAD⁺. Approximately 3.5 moles of sulfhydryl groups per enzyme reacted with DTNB in the presence of NAD⁺ but the modified enzyme still retained 70% of its original activity. The addition of sodium dodecylsulfate caused further reaction of the enzyme with DTNB and 8moles of sulfhydryl groups per enzyme were detected in total by the DTNB method.

View full abstract

Two kinds of C-type cytochromes, cytochrome c-552 and cytochrome c-554, derived from the chemoautotropfa, Nitrosomonas europaea, were purified to an electrophoretically homogeneous state and their properties determined. Cytochrome c-552 possessed absorption peaks at 410nm in the oxidized form, and at 416, 523, and 552nm in the reduced form. The isoelectric point of the cytochrome was at pH 3.7, its midpoint redox potential was about +0.25V and its molecular weight about 10, 000. The cytochrome reacted rapidly with Pseudomonas aeruginosa nitrite reductase [EC 1. 9. 3. 2] and at an appreciable rate with a cytochrome oxidase preparation derived from N. europaea, but did not react either with cow cytochrome oxidase [EC 1. 9. 3. 1] or with yeast cytochrome c peroxidase [EC 1. 11. 1. 5].
Cytochrome c-554 had absorption peaks at 407nm in the oxidized form, and at 421, 524, and 554nm in the reduced form. The isoelectric point of cytochrome c-554 was at pH 10.7. The molecular weight of this cytochrome was 10, 700 on the basis of the heme content and dry weight, while it was found to be 21, 500 by gel filtration with a Sephadex G-100 column and 25, 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Cytochrome c-554 was autoxidizable.
Hydroxylamine-cytochrome c reductase (hydroxylamine: cytochrome c oxidoreductase, [EC 1. 7. 3. 4]) of N. europaea did not react with cytochrome c-552, while it reduced the cytochrome with hydroxylamine in the presence of cytochrome c-554. On the basis of these facts, together with the reactivity of cytochrome c-552 with N. europaea cytochrome oxidase, the electron transfer mechanisms coupled to the oxidation of hydroxylamine in the organism were discussed. The evolutionary position of N. europaea was also discussed on the basis of the enzymatic properties of cvtochrome c-552.

View full abstract

Studies were made on the amino acid residue of myosin A which when modified with diazobenzene-p-sulfonate (DBS), resulted in loss of the EDTA-ATPase activity of myosin A ATPase [EC 3. 6. 1. 3]. A cysteinyl residue(s) seemed to be responsible for this change in catalytic properties, judging from the results of spectrophotometric studies, determination of residual cysteine after modification with DBS by carboxymethylation with ¹⁴C-monoiodoacetate and amino acid analysis. ¹⁴C-Carboxymethyl-myosin A was digested with trypsin [EC 3. 4. 21. 4] before and after modification with DBS and radioautograms of SH-peptide maps of four fractions obtained by gel filtration from the digests, reveal that the sulfhydryl group which was modified with DBS was not identical with the sulfhydryl groups, S₁ and S₂, in the active site of myosin, which can react with N-ethylmaleimide. It was proposed that this sulfhydryl group is named S₃.

View full abstract

ω-Heparin isolated from whale organs was digested with an eliminase [heparin lyase, EC 4. 2. 2. 7] prepared from crude heparinase, which was obtained from α-heparinadapted flavobacteria. The digestion products were then fractionated into 16 fractions by ion-exchange column chromatography on Dowex-1. Subsequently, the resulting fractions were purified by rechromatography on Dowex-1 and/or by preparative paper chromatography, yielding almost quantitatively purified compounds in fractions 2, 4, 5, 6, and 12. In addition, compounds lOa and 15a were isolated in a homogeneous state from fractions 10 and 15.
Compounds 2, 4, 5, 6, lOa, 12, and 15a were characterized and identified as N-acetylglucosamine, Δ4, 5-hexosyluronic acid-N-acetylglucosamine, N-sulfated glucosamine, Δ4, 5-hexosyluronic acid-N-sulfated glucosamine, N-, 6-O-disuIfated glucosamine, and most probably sulfated Δ4, 5-hexosyluronic acid-N-sulfated glucosamine and sulfated Δ4, 5-hexosyluronic acid-N-, 6-O-disulfated glucosamine, respectively, by chemical and enzymic studies together with infrared and ultraviolet spectral analyses. Furthermore, the major fractions (fractions 12, 13, 14, and 15) were each shown to contain two or three sulfate residues per unsaturated disaccharide unit.
The total yield of unsaturated oligosaccharides was 66.6% of the starting material, while the major fractions accounted for 48.2%.
In agreement with previous data, the major glucosaminidic linkages of ω-heparin appear to be 1→4.
The present study suggests that ω-heparin has a more complex structure than α-heparin.

View full abstract

A new phospholipid, isolated from extracts of Cryptococcus neoformans, has beert identified as pyrophosphatidic acid. Acetolysis of the lipid gave diglyceride-monoacetate amounting to 98% of the original weight. Analysis of the lipid gave the mole ratio 1:1 for diglyceride-phosphorus. Quantitative degradation of the lipid with aqueous pyridine yielded phosphatidic acid. This result suggests the presence of a pyrophosphate bond in the molecular structure. Spectrometric (IR, ³¹P-NMR) analyses also showed the presence of a P-O-P bond. The sodium form of the lipid contained 1 mole of sodium per mole of phosphorus, and the amount of alkali necessary to neutralize the acidic groups of the free form of the lipid corresponded to one-half that for phosphatidic acid.
Furthermore, the results of these chromatographic, chemical, and spectrometric analyses agreed fairly with those for chemically synthesized pyrophosphatidic acid.
The fatty acid composition of pyrophosphatidic acid from yeast was C_16:0 acid (33%), Cieu (33%), and C_18:2 (34%), being very similar to the composition of phosphatidic acid but different from that of cardiolipin.

View full abstract

A radioimmunoassay for phenobarbital has been investigated. Antiphenobarbital antisera were obtained by single immunization or repeated immunization of rabbits with p-azophenobarbital protein. The conditions for radioimmunoassay using ¹⁴Cphenobarbital (specific activity 3.15mCi/mmole) and ³H-phenobarbital (specific activity 10.3Ci/mmole) are described. Eighty picograms of phenobarbital could be detected by this technique. The determination of serum phenobarbital concentration in the mouse after intraperitoneal administration was carried out.

View full abstract

Choline and ethanolamine phosphoglyceride fractions were isolated from the lipid extract of rabbit sarcoplasmic reticulum. The fatty chains of the diacyl, 1-O-alkenyl2-acyl and l-O-alkyl-2-acyl components were analyzed by selective acidic, alkaline, and enzymatic hydrolyses.
Similar compositions of fatty chains at the 1-and 2-positions of l-O-alkenyl-2-acyl and l-O-alkyl-2-acyl components were observed, whereas significant differences were found between the diacyl and the other two components in ethanolamine phosphoglycerides. For choline phosphoglycerides, no structural relationship between the diacyl and the other two components could be definitely determined.
These findings suggest that a metabolic relationship between the 1-0-alkenyl and 1-O-alkyl moieties may exist and, in the ethanolamine fraction, an intimate relationship of the diacyl component with ether-type phosphoglycerides can be excluded.

View full abstract

1. Phosphatidylcholine and sphingomyelin, which accounted for about 60 percent of the total phospholipids of brain microsomes, were completely hydrolyzed by phospholipase C [EC 3. 1. 4. 3] treatment.
2. The amount of bound Mg²⁺ in microsomes treated with phospholipase C was 1.3-1.5 times as much as that in non-treated microsomes. This fact indicated that the phosphate groups of phospholipids are not the major cation-binding sites. It is suggested that, as a result of this treatment, cation-binding sites which were buried became exposed at the surface of membranes.
3. On treatment of microsomes with neuraminidase [EC 3. 2. 1. 18], almost all sialic acids of brain microsomes were removed, but Mg²⁺-binding capacity did not change.
4. To investigate the role of carboxyl groups in microsomal protein as binding sites, a water-soluble carbodiimide, l-ethyl-3 (3-morpholinyl-(4)-propyl) carbodiimide (EMPC^*), was used to modify the free carboxyl groups. The degree of modification was estimated by amino acid analysis, which gave the amount of glycine incorporation into microsomes.
5. As the amount of glycine incorporation increased from 0 to 240nEq/mg protein, the capacity for cation binding decreased from 540 to 60nEq/mg protein at pH 7.5. Thus, it is concluded that the carboxyl groups on the side chains of aspartic acid and glutamic acid in microsomal protein provide the major sites of ion exchange-type cation binding in brain microsomes.

View full abstract

The effects of glucose and cAMP on the aggregation of D. discoideum were studied. The number of aggregation centers increased and the duration of the aggregation stage was prolonged by the addition of glucose, during the initial 2hr after the deprivation of food. This effect paralleled the uptake of glucose by this organism. The number of aggregates increased drastically on addition of external cAMP as aggregation proceeded, and the uptake of cAMP by the cells decreased. The duration of the aggregation stage was also prolonged by added cAMP. The effects of glucose and cAMP on the early development of D. discoideum are discussed.

View full abstract

Adrenaline-sensitive lipid micelles were prepared from rat epididymal adipose tissue by hypotonic treatment. The lipolytic activity (free fatty acid release) of these micelles was stimulated by DBcAMP, theophylline and caffeine, but not by cyclic 3', 5'-adenosine monophosphate (cyclic AMP), adenosine or adenosine monophosphate.
Lipolysis in lipid micelles was also increased by phospholipase C [EC 3. 1. 4. 3]
but not by phospholipase D [EC 3. 1. 4. 4].
When adrenaline and phospholipase C were incubated together with the lipid micelles, their effects were not additive.
The lipolytic activity of lipid micelles prepared from isolated fat cells which had been preincubated with 10μg per ml of adrenaline was higher than that of micelles preincubated without adrenaline and was not stimulated by further addition of adrenaline to the assay system.

View full abstract

The binding and spectral properties of bromocresol purple (BCP), an acidic dye, were used as probe to investigate structural features of fragmented sarcoplasmic reticulum (SR) of skeletal muscle.
The wavelengths of maximum absorption intensity (λ_max) of BCP were 589 and 604nm in Tris-HCl buffer (pH 8.8) and bound to SR, respectively. Since spectral shift of BCP to longer wavelengths (red shift) is caused by approach of the hydrophobic radical of an adjacent molecule, it was suggested that the red shift of BCP on SR reflected the hydrophobic nature of SR.
When the Ca²⁺ concentration was less than 10^-9M, ATP released BCP molecules from SR. The resulting increase of free BCP concentration in the reaction mixture shifted the total spectrum to shorter wavelengths (blue shift), though λ_max of the bound BCP did not change. The release of BCP after ATP addition was interpreted as a result of increase in the number of nucleophilic sites on SR. If Ca²⁺ was present, ATP addition enhanced the binding of BCP and caused a marked decrease in the absorption intensity of bound BCP, suggesting a decrease in the number of nucleophilic sites on SR during Ca²⁺ transport.
The blue shift caused by release of BCP was used to estimate the interaction of ATP with SR using a dual-wavelength spectrophotometer, yielding association constants of ATP for SR of 1.2×l0⁵ and 2.5×lO⁴M^-1 in the presence and absence of Mg, respectively.

View full abstract

The catabolic activity of hemolytic streptococci for nucleosides was examined usingculture supernatant, resting cell systems, toluenized cells or cell-free extracts. Although exoenzymes and exotoxins such as endonuclease or streptolysins were present, nucleoside-catabolic enzymes were not detected in the culture supernatant. Phosphorolytic cleavage of thymidine, uridine, and deoxyguanosine was demonstrated in resting cells as well as in cell-free extracts. The rate of nucleoside degradation in washed streptococci was considerably enhanced by toluenization. Trans (deoxy)-ribosylation between thymidine (or uridine) and adenine occurred in resting cell systems but not in cell-free extracts. Deoxyribosyl-transfer reaction was observed in a cell-free system which contained purine-nucleoside phosphorylase [EC 2. 4. 2. 2] from hemolytic streptococci and thymidine phosphorylase [EC 2. 4. 2. 4] from E. coli. Cell-free extracts from streptococci were deficient in deaminase activities for (deoxy)-cytidine, cytosine, or adenosine. (Deoxy)cytidine was converted to uracil but cytosine was not deaminated, upon incubation with resting cells. The requirements for nucleoside catabolism in resting streptococcal cells were examined.

View full abstract

Phosphorylation of deoxyribonucleosides in relation to DNA synthesis was investigated with embryos of the sea urchins, Hemicentrotus pulcherrimus and Anthoddaris crassispina.
Thymidine added to the medium was taken up rapidly into the acid-soluble fraction of embryos followed by phosphorylation, accumulating more than 80% as thymidine triphosphate. The phosphorylations of thymidine did not fluctuate in parallel with thymidine incorporation into the DNA fraction. Thymidine triphosphate derived from exogenously added thymidine was some 10-fold larger than its amount polymerized to DNA. The percentage incorporation of thymidine triphosphate into the DNA fraction, measured in vivo and the DNA polymerase [EC 2. 7. 7. 7] activity assayed in vitro, both varied in parallel with thymidine incorporation into the DNA fraction during the cell cycle, while thymidine kinase [EC 2. 7. 1. 75] and thymidylate kinase [EC 2. 7. 4. 9] activities assayed chromatographically did not. DNase [EC 3. 1. 4. 5] activity measured with homogenates also did not fluctuate during the cell cycle.
The phosphorylations of deoxyadenosine, deoxycytidine, and deoxyguanosine were somewhat different from that of thymidine, but, in general, the amount of triphosphate derivatives originated from exogenously added deoxyribonucleosides was much larger than that incorporated into the DNA fraction.

View full abstract

1. Chromatophores from Rhodospirillum rubrum exhibited two distinct ESR signals of g=2.0 and g=4.3. On addition of either succinate or NADH, these two signals decreased, while a new signal of g=1.93 appeared. The signals of g=1.93 formed by succinate and by NADH were not additive in intensity, and were indistinguishable in shape.
2. The signal of g=2.0 was due to organic free radicals. Most of the signal intensity was due to the semiquinone form of quinone bound to the Chromatophores.
3. Of the three signals, the signal of g=1.93 was studied most extensively. The E_{m, 7} value of the component responsible for the signal was +0.04V with n=1. The reduction of the component was not influenced by extraction of the bound quinones.

View full abstract