The incorporation of P³² into the phospholipid and the residual fractions of avian tuberble bacilli was studied under various conditions. By the addition of inorganic phosphate and under anaerobic condition, the incorporation rate of P³² was reduced. The incorporation rate increased after treating the bacilli in veronal buffer for 4 hours at 37°.
The effect of various inhibitors, cofactors, antibiotics metal ions upon P³²-incorporation was also investigated and the mechanisms of the process were discussed.
This work was carried out under the direction of Prof. K. Hoshishima to whom the author wishes to express his thanks for the constant guidance in the course of this work.
I wish to express my sincere gratitude to Dr. K. Matsui, National Sanatorium Toneyama Hospital, for supplying us the avian tubercle bacillus, strain Takeo. I am also indebted to Dr. T. Shirotori, Toa Alimentary Chem. Ind. Co., Ltd., for his kindness to furnish us a sample of FAD.
Thanks are due to Prof. M. Umezu and Prof. T. Uemura, Faculty of Agriculture of Tohoku University, Sendai, and to Prof R. M. C. Dawson, Biochemistry Department, A. R. C. Institute of Animal Physiology, Babraham, Cambridge, England, for their invaluable advice and criticism.

View full abstract

1. Hydrolysis of benzoyl-Arg-NH₂ and the activity of trypsinogen-activa-tion by the trypsinogen-kinase and other various proteases were examined. Both the trypsinogen-kinase (acid-protease from Asp. oryzae) and the acid-protease from Asp. saitoi could not hydrolyse benzoyl-Arg-NH₂ but activated trypsinogen strongly.
2. The neutral-protease from Asp. oryzae has also a weak activity in trypsinogen-activation.
3. The preparation of the author's acid-protease has the activities of carboxypeptidase A, B, proteinase, and of hydrolysing L-Leu-L-Arg and L-Phe-L-Arg.
4. The relation between the substrate specificity of the acid-protease from Asp. oryzae and the mechanism of trypsinogen-activation by this enzyme are discussed.
The author wishes to express his thanks to Prof. Akabori of Osaka University for his kind guidance, and to Mr. Nomoto, Mr. Miura and Dr. Yoshida for supplying the used proteases, and to the staff of the Ando Laboratory of Tokyo University for giving the arginine containing peptides.

View full abstract

Enzymatic synthesis of cocarboxylase in rat liver preparation was studied. For this reaction the enzyme requires thiamine as a phosphate acceptor, ATP as a phosphate donor, and divalent cation(s), Mg⁺⁺ or Mn⁺⁺, as an activator. Among the adenine nucleotides tested only ATP could serve as a direct substrate and other nucleotides, such as ADP and A5P, did not act as a phosphate donor but behaved as an inhibitor. TMP did not act as a phosphate acceptor but behaved also as an inhibitor. The effectiveness of ADP and A5P+PEP in the crude system may be interpreted as the result of intermediary formation of ATP brought about by other enzymes. Thus, it could be concluded that the only possible mechanism for the reaction is a one-step transpyrophosphorylation. The reaction could not be reversed easily starting from TDP and A5P. The Michaelis constants for thiamine and ATP were calculated as 4.5×10^-7 M and 8.3×10^-4 M, respectively. The enzyme was inhibited by ethylenediamine tetraacetate.
The author express his sincere thanks to Prof. N. Shimazono for guidance and encouragement during the course of this investigation.

View full abstract

The RNP was isolated from the liver microsomes of adult rat and the effect of urea on the dissociation and on the RNase activity of the RNP were examined.
1. Electrophoretic analysis showed that the RNP was hardly dissociated to free RNA and protein at pH 7.0 even in the concentrated urea solution, though it was gradually degraded to a slower component (component 3), of which RNA and protein are probably linked loosely by ionic bonds. This estimation is also supported by centrifugal analysis.
2. Paperchromatography in urea succeeded to demonstrate the dissociation of the RNP. When the RNP was developed in the concentrated urea solution, protein stained with bromphenol blue was always found at or near the starting line, while most RNA was detected near the solvent front. Eluted solutions from the paper, however, showed that this RNA contained considerable amount of Folin positive substances.
3. Either the autodegradation of the RNP and the RNase activity of the RNP were slightly activated by urea.
This work was supported by grants from the Wacksman Foundation in Japan.

View full abstract

1. The inhibition of glycylglycine dipeptidase of yeast by various amino acids was investigated kinetically. The inhibition caused by L-leucine was competitive with substrate but not with Co⁺⁺ ion. A scheme for the inhibition produced by amino acid was proposed which was shown to satisfactorily agree with the experimental results.
2. The inhibitory activities of various amino acids were compared in terms of the affinity constants of these substances towards the enzyme.
The author wishes to express his sincere gratitude to Prof. A. Takamiya for his valuable advices in this work.

View full abstract

EDTA and six analogues were tested for their relaxing action on glycerinated psoas muscle fibers, their potentiating effect on myosin ATPase and their synergistic action with inorganic polyorthophosphate on glycerinated fibers.
1. The relaxing action of the chelating compounds was shown to have any quantitative correlation with their chelating action on neither Ca nor Mg. GEDTA, which has practically no chelating capacity for Mg under the experimental conditions, showed a remarkable relaxing effect, several times as strong as EDTA.
2. The potentiating action of the chelating agents on myosin-A ATPase has no correlation with their chelating effect on Ca, but showed some correlation with their chelating capacity for Mg.
3. The lengthening of glycerinated muscle fibers by inorganic polyorthophosphate was markedly accelerated by EDTA and its analogues; thus elongated fibers were found without contractility. This action did not parallel the chelating activity.
4. The abovementioned three actions of chelating agents showed no correlation with one other.
The authors wish to express their thanks to Prof. H. Kumagai for his encouragement and to Dr. S. Watanabe at the Dartmouth Medical School for his discussions.

View full abstract

After administration of p-aminobenzoic acid to human beings, dogs, rabbits and mouse, metabolic products contained in urines were identified paper-chromatographically. When p-aminobenzoic acid and glucose was administered simultaneously to a rabbit, N-glucoside combined to three amines (p-aminobenzoic acid, p-aminohippuric acid and p-aminobenzoyl glucuronide) were detected in the urine.
We wish to thank Prof. H. Yoshikawa for his kind advice and constant leading.

View full abstract

The acid-soluble extract of Tetrahymena geleii W was analyzed by column chromatography. The identification was based on ultraviolet absorption spectra and chromatographic methods, together with chemical and isotopic techniques.
Evidence is given for the presence of 5'-monophosphates of adenosine, guanosine, uridine, and cytidine besides ADP, ATP, UTP, and DPN. In addition, the existence of unidentified phosphate compound which showed sugar reaction was presented.
The natural occurrence of 2'- and 3'-mononucleotides was presented and discussed.

View full abstract

“Fourth flavin compound”, which had been found in heated liver homogenate, was formed from purified FAD by heating its aqueous solution. The conditions for optimum formation of FFC from FAD were found as follows:
1. Optimum pH existed at between pH 4 and 8.
2. FFC formation was increased with ascending temperature until 130°.
3. The formation of FFC decreased with increasing concentration of FAD.
Under the optimum conditions, FFC was produced from FAD, purified and crystallized.

View full abstract

1. N-Terminal sequences of two protamines, salmine from salmon (Oncorhynchus keta) and clupeine from herring (Clupea pallasii), were studied. For that purpose, DNP-protamines were hydrolyzed by trypsin and the DNP-peptides in the dried digests were extracted with methanol. The methanol-soluble DNP-peptides were fractionated into two parts by continuous paper electrophoresis, one (designated as DNP-band A) with one arginine residue and another (DNP-band B) with two. Each of the DNP-bands A and B was purified and fractionated by running through a talc column and the structure of the isolated DNP-peptides was determined.
2. Since only two DNP-peptides, DNP-Pro•Arg and DNP-Pro•Arg•Arg, were obtained from DNP-salmine, the N-terminal sequence of salmine was inferred to be exclusively Pro•Arg•Arg•••.
3. On the other hand, four DNP-peptides, DNP-Ala•Arg, DNP-Pro•Arg, DNP-Ala•Arg•Arg and DNP-Pro•Arg•Arg, were isolated from DNP-clupeine, thus the N-terminal structure of clupeine was concluded to consist of two separate chains, Ala•Arg•Arg••• and Pro•Arg•Arg The mixed ratio of the both chains was also estimated.
The authors' thanks are due to Dr. K. Imahori, Department of Chemistry, College of General Education of this University, for his valuable advice and discussion concerning the application of paper electrophoretic technique to this study. The present work was supported in part by a Scientific Research Grant from the Ministry of Education.

View full abstract

The process of connective tissue growth in granuloma induced by the subcutaneous injection of alginate into rats was investigated over 100 day period.
Differently from the carrageenin-granuloma reported by Jackson et al., the development of new connective tissue was quite slowly achieved and the reabsorption of collagen fibers was scarcely recognized throughout the experimental period.
The relative amount of aminopolysaccharides associated with developing collagen fibers decreased with maturing or aging of fibers.
The administration of ascorbic acid accelerated considerably the reaction from proline to hydroxyproline, i.e. the formation of collagen fibers.

View full abstract

1. l-Alkoxynicotinamide and related compounds were synthesized as model compounds of pyridine coenzymes. Cyanide addition reaction to these compounds was studied and the resulting cyanonicotinamides and their derivatives were isolated.
2. The ratio of 4- and 6-cyanonicotinamide formed was measured by the weight of dimethyl pyridine-dicarboxylates. Addition at 4-position was favored by a high dielectric constant and a rather low pH value of the solvent and model compounds with large l-substituents.
3. l-Alkoxy-3-carbonylpyridinium compounds were found to have a marked absorption near 380 mμ in alkaline solution. This absorption vanished during the course of cyanide addition reaction owing to spontaneous decomposition of the cyanide addition product into cyanonicotinamide and alcohol. The rate of disappearance of the absorption in the reaction decreased at a lower KCN concentration, in strong alkali, and at lower dielectric constant.
4. Selective addition at 4-position and light absorption near 340 mμ of pyridine coenzymes which observed in the addition reaction are discussed.
The writer expresses thanks to Prof. T. Okamoto for suggesting this investigation as well as for constant guidance in the course of the work, and to many courtesies extended to him during this work in the Department of Pharmaceutical Chemistry. The writer is indebted to Mr. H. Tani for gift of many samples. Elementary analysis was kindly performed by members of the Central Analysis Department. The writer also ex-presses his heartful gratitude to Prof. N. Shimazono and Prof. E. Ochiai for their helpful advices and continuous encouragement.

View full abstract

Conchiolin, a scleroprotein obtained from the shell of the pearl oyster (Pinctada martensii), was fractionated with 70 per cent acetic acid, 2 N ammonia, the concentrated solution of copper-ethylenediamine, and [Co (py)₄] Cl₂ in water, respectively. By these treatments, several fractions of conchiolin were obtained.
The N-terminal amino acids of these fractions of conchiolin were proved to be glycine, serine and aspartic acid. The C-terminal amino acids of Conchiolin I-l (70 per cent acetic acid and 2 N ammonia soluble conchiolin) were glycine, serine and threonine.

View full abstract

1. Using the in vitro method and S³⁵ DL-methionine, we tested the rate of amino acid transfer from the mucosa to the serosa of rat intestine and vice versa. The transfer rate of the L-isomer from mucosa to serosa was shown to be much faster than that of the D-isomer. However, when the same experiments were carried out from serosa to mucosa, there was no difference between the L- and D-isomer. In this case, the rate of transfer was reduced to that of free diffusion.
2. In B₆ deficiency induced by L-penicillamine in vivo, the rate of absorption of the L-isomer is decreased and restored by B₆ to the normal rate. No acceleration of the rate of absorption was observable with the D-isomer. The effect of B₆ was not duplicated by other vitamins such as B₁ or B₂. The absorption of glucose under the same experimental conditions was not influenced by B₆. Furthermore, the rate of L-methionine absorption was not affected in B₁ deficiency.
3. Evidence is given that the animals after injection of L-penicillamine were in B₆ deficiency.

View full abstract

1. Observation was made on the metabolic pattern of amino acids in the intestinal wall and portal blood after absorption of S³⁵ DL-methionine and C¹⁴ DL-valine.
2. Amino acids after entering the intestinal tissues seem to be independent of the pool of amino acids in the tissues.
3. About half the methionine observed was oxidized to methionine-sulfoxide in the intestine, and was again reduced in the portal circulation.
4. The above oxidoreduction of methionine appears to be coupled with its mechanism of absorption.
5. C¹⁴ DL-valine enters the portal blood without any modification.

View full abstract

1. The interference between L-valine, L-leucine and L-isoleucine during absorption from the intestine was studied both in in vivo and in vitro.
2. These three amino acids have a characteristic competitive action on the absorption mechanism. The interference between other amino acids such as methionine and histidine is not reciprocal.
3. Competition may occur in the earlier stage of absorption and be related to the step of active transportation.

View full abstract