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1. 1. Lymph nodes of hyperimmunized rabbits have been found to contain polyribosomes which are dissociated into monomers by ribonuclease. 2. 2. By means of an enzyme-antienzyme system, specific inhibition of bacterial α-amylase activity with these ribosomal fractions has been observed.

It was shown that a single intraperitoneal injection of extract (fraction 3 and 4) from roots of Panax ginseng C. A. MEYER, increased activity of RNA polymerase (EC 2.7.7.6), assayed in vitro, of deoxycholate-lysed nuclei of rat liver. The maximum increase of the enzyme activity was found at 2 hr with a lag period of 30 min after treatment. The increase was still observed 8 hr after the injection of the extract but disappeared after 16 hr. The increase in the activity was found in the presence of high concentration of ammonium sulfate in vitro as well as in the absence of the salt.Three hours after treatment in vivo, a little increase of protein synthesis was also observed in liver homogenate or nuclei and abolished by prior administration of puromycin. The increase in the activity of the polymerase due to ginseng extract did not disappear even when the synthesis of bulk nuclear protein was greatly inhibited by puromycin.

In the course of our study on kynurenic acid metabolism we observed that very concentrated sonic extracts of a pseudomonad, employing the so-called “quinoline pathway”, were able to degrade D-tryptophan. In this communication we wish to describe a new enzyme system which catalyzes the conversion of D-tryptophan to D-kynurenine, together with a new enzyme, D-kynurenine oxidase, which catalyzes the oxidative deamination of D-kynurenine to kynurenic acid. Recently Behrman and Cullen (1961) found tryptophan racemase in another strain of pseudomonad, which metabolizes D-tryptophan via the so-called “aromatic pathway”, but in our strain of pseudomonad D-tryptophan is metabolized without racemization to the L-isomer.

1. V. B1欠乏飼料を投与したラットは, はじめ2週間までは体重はやや増加したがその後欠乏日数の経過と共に減少し, 31日では初体重よりも減少した。 しかしV. B1投与によって回復した。2. V. B1欠乏ラットの湿肝重量は欠乏18日までは, わずかに増加するがその後増加は認められなかった。3. 肝細胞におけるV. B1はその大半が細胞質に存在し欠乏日数の経過と共に急速に減少した。4. ポストミトコンドリヤ上清画分のポリソームパターンは, 欠乏18日よりheavy polysomesの減少, lightpolysomesの増加を示し, この傾向は欠乏25日および31日では更に顕著にあらわれ, V. B1投与後は急速に回復した。5. 精製ポリソームパターンにおいては欠乏7日のポリソームは, 対照に対してheavy polysomesの増加の傾向がみられ, 3H-オロチン酸の取り込みもtrimer以上のポリマー部分において増加した。 欠乏25日および31日ではheavy polysomesが減少しlight polysomesの増加が顕著にあらわれ, 3H-オロチン酸の取り込みも極度に減少した。 なおhexamer以上のポリソームの全リボソームに対する割合は, 欠乏7日で一過性の増加を示しその後減少した。6. in vivoにおける3H-オロチン酸のポリソームRNAへの取り込みの比活性, およびin vitroにおける14C-ロイシンの取り込みの比活性は, いずれも欠乏7日に一過性の増加を示し, その後欠乏日数の経過と共に減少した。 またin vivoにおける核RNAへの3H-オロチン酸の取り込みの比活性は欠乏7日で対照よりやや増加しているが大差は示さず, 欠乏18日より減少した。 そしていずれもV. B1投与後は急速に回復した。

Partial hepatectomy in the rat is known to cause rises in the rates of incorporation by subcellular liver preparations of 3H-puromycin into nascent peptide and 14C-leucine into protein. The operation has also been shown to be followed by a rise in the rate of liver protein synthesis in vivo. The kinetics of the three rises after partial hepatectomy are the same, suggesting that the changes are interrelated. The effects of actinomycin D on the rises were studied. When the antibiotic (5 µg per hour) was given immediately after partial hepatectomy, it blocked the appearance of the rises in vitro and in vivo and, as a function of dose, affected the three changes in an indistinguishable manner. When actinomycin (10 µg per hour) was given to partially hepatectomized animals after the rises had occurred, the reactivities in vitro and in vivo were gradually reduced to the normal levels. The halfway points in the return to the normal levels were at 4 hours from the time of the first injection of the antibiotic.

Intraperitoneal administration of ginseng extract (fraction 3 or 4) to rat was found to increase the rate of synthesis of serum proteins such as albumin and γ-globulin. The incorporation rate of 3H-leucine into serum proteins was significantly increased 4 hr after the injection of fraction 3. The increased rate of protein synthesis reached a maximum about 8-12 hr after the administration and 46-49% increase in the rate of serum protein synthesis was observed. It was confirmed by immunochemical precipitation reaction that the increase in serum protein synthesis was due to the stimulation of albumin and γ-globulin synthesis. The rate declined to the control level at 20 hr following ginseng extract treatment. These observations are similar to the effect of cortisone on the liver. Although the mechanism of this increase in the rate of serum protein synthesis by ginseng extract is not known, some differences were observed in effects between fraction 4 and cortisone as follows. Induction of tryptophan pyrrolase and tyrosine transaminase, which is observed with cortisone, did not occur by fraction 4 administration. These results suggest that the observed effect of ginseng extract would not be due to the elevated concentration of adrenocortical steroids. From this point of view, this stimulating factor, which we term "prostisol"(protein synthesis stimulating factor), is active to RNA and protein syntheses when administered in vivo.

Partial hepatectomy and acute stress (intraperitoneal injection of Celite) cause increases in the rates of synthesis of serum albumin and fibrinogen, proteins that are formed mainly or entirely by the liver. The rises in protein synthesis after removal of part of the liver differ kinetically from those after acute stress. In addition, only the response to partial hepatectomy is independent of the adrenal glands. Both responses seem to be related to an increase in the ability of liver polyribosomes to form protein.

The initial rates of incorporation of amino acids by liver microsomes and ribosomes (prepared without detergent and with polyuridylic acid in place of endogenous messenger RNA) from normal, partially hepatectomized, and acutely stressed (Celite-treated) rats are linear. Removal of part of the liver and stress cause increases in the initial rates of incorporation. The reactivities of liver microsomes and ribosomes with puromycin are also enhanced by partial hepatectomy and acute stress. The enhanced rates of reaction with the antibiotic were observed in the presence and absence of guanosine triphosphate and over a wide range of Mg++ concentrations. The changes in reactivity with puromycin parallel the changes in vitro and in vivo in liver protein synthesis as a function of time after partial hepatectomy and acute stress. When normal liver microsomes are treated with 1% deoxycholate, the initial rates of incorporation of 14C-leucine and 3H-puromycin are raised to the levels ordinarily found with untreated preparations from partially hepatectomized and stressed animals. The initial rates with preparations from partially hepatectomized and stressed animals are not affected by the detergent. Similar results are obtained when ribosomes have been treated with deoxycholate. These results suggest that liver ribosomes contain a factor that regulates peptide bond synthesis or the attachment of aminoacyl transfer RNA. Removal of part of the liver and acute stress may reduce the function of the regulatory factor, and deoxycholate may remove it from the ribosome.

The in vivo administration of extract of the roots of Panax ginseng C. A. MEYER increased the incorporation rate of labeled precursors into liver nuclear RNA at 4 hr and into cytoplasmic polysomal RNA at 5.5-6.0 hr after a single dose intraperitoneal injection in a rat. The incorporation rate was directly dependent on the amount of a fraction obtained by partial purification. The increased specific radioactivity of the liver RNA is not due to the change in the pool size of endogenous acid-soluble nucleotides.

Tryptophan pyrrolase ( L -tryptophan: H 2 O 2 oxidoreductase, EC 1.11.1.4) and tyrosine-α-ketoglutarate transaminase ( L -tyrosine: 2-oxoglutarate amino transferase, EC 2.6.1.5) activities of rat liver homogenate were increased about 2–2.5 fold in 4–6 h after laparotomy, and 4–6 fold in 8–10 h after partial hepatectomy, without loading of gluccorticoid or substrate. The rise of tryptophan pyrrolase was inhibited by actinomycin D administered immediately after operation. In adrenalectomized rats, the induced tryptophan pyrrolase activity was inhibited. The increased activity in tryptophan pyrrolase after partial hepatectomy has been attributed to change in supernatant fraction, and was not affected by administration of cortisone.

Publisher Summary This chapter discusses the methods of preparation of D -Amino Acid Dehydrogenase (Pseudomonas fluorescens). D -Amino acid dehydrogenases of Pseudomonas fluorescens catalyze the oxidation of D -amino acids in the presence of an appropriate hydrogen acceptor, liberating ammonia. Two distinct D -amino acid dehydrogenases, each showing absolute specificity for methylene blue or 2,6-dichloroindophenol, respectively, have been isolated. The methylene blue-specific D-amino acid dehydrogenase was detectable only in extracts from D -tryptophan-grown cells. The 2,6-dichloroindophenol- specific D -amino acid dehydrogenase is constitutive, being present in all cell extracts irrespective of culture conditions. D -Kynurenine was oxidized to kynurenic acid. Residual kynurenine was measured by the Bratton-Marshall procedure. A unit of enzyme was defined as the amount that caused the disappearance of 1 micromole of D -kynurenine per minute. Specific activity was expressed as units per milligram of protein. Under the conditions of the assay, the reaction rate was proportional to the amount of enzyme (up to 0.18 unit of enzyme) and the reaction rates were linear with time during the periods employed.

OBSERVATIONS made by Okazaki et al.1,2 and Oishi3 support the idea that relatively small polydeoxynucleotides are intermediates in the replication of strands of both bacterial and phage DNA. The polydeoxynucleotides are subsequently joined to each other and to the growing DNA strands. This mechanism would allow a single kind of DNA polymerase to copy both parental DNA strands in the 5′ to 3′ direction.

Partial hepatectomy and acute stress (intraperitoneal injection of Celite) caused a decrease in the activity of serum ribonuclease. The decrease in activity after partial hepatectomy differed kinetically from that after acute stress. The decreased activity of serum ribonuclease was closely related to an increase in the level of ribonuclease inhibitor in the serum.

Partial hepatectomy and acute stress (intraperitoneal injection of celite) caused an increase in the activity of serum deoxyribonuclease inhibitor. The increased activity after partial hepatectomy differed kinetically from that after acute stress. Deoxyribonuclease inhibitor was purified from rat liver about 30-fold and its properties were studied and found to be those of a protein. The activity of the inhibitor in rat liver increased after partial hepatectomy. The activity of the inhibitor may not be related to the activity of DNA synthesis as assayed by the incorporation of [ 3 H]thymidine.

Polynucleotide ligase, which catalyzes the covalent joining of two segments of an interrupted strand in a DNA duplex, was extracted from rat liver. The activity is located in both nuclei and soluble fractions. Its optimal pH was 8.0. The enzyme requires ATP as a cofactor. Its activity was completely dependent on the presence of Mg 2+ . The activities of these enzymes in regenerating rat liver were 4 times higher, in both fractions, than those in normal rat livers.

It was shown that a single intraperitoneal injection of extract (fraction 4) from roots Panax ginseng C.A.Meyer, increased the incorporation rate of labeled precursor into cytoplasmic polysomal ribonucleic acid (RNA) of rat liver.The content of heavy poly-somes (over hexamer) in postmitochondrial supernatant from liver treated with fraction 4 was observed to increase at 6 and 10 hr after treatment.Sedimentation analysis of ribosomal RNA and messenger RNA derived from polysomes showed that the newly induced RNA by fraction 4 is evenly distributed over all RNA. Particularly, 29S ribosomal and 9-10S messenger RNAs were stimulated significantly. In an amino acid incorporation system in vitro, stimulatory activities of microsomes and polysomes from treated rat liver are more active by 85 and 67% than those from normal liver, respectively.

Rat serum ribonuclease has been purified about 6000-fold. The optimal pH of this enzyme was 7–8. The purified enzyme was rather heat labile at pH 7.6. The Km of this enzyme is 30% higher than that of the crystalline bovine pancreatic ribonuclease (polyribonucleotide 2-oligonucleotide-transferase, EC 2.7.7.16). No stimulatory or inhibitory effect on this enzyme was observed with EDTA, KCl, MgCl2. p-chloromercuribenzoinc acid, or mercaptoethanol.

Two distinct d-amino acid dehydrogenases, each showing absolute specificity for methylene blue or 2,6-dichloroindophenol, respectively, were isolated from Pseudomonas fluorescens (ATCC 11299B). The methylene blue-specific d-amino acid dehydrogenase was detectable only in extracts from d-tryptophan-grown cells and was purified about 40-fold. The 2,6-dichloroindophenol-specific d-amino acid dehydrogenase is constitutive, being present in all cell extracts irrespective of culture conditions, and it was purified about 65-fold. These enzymes appear to be flavoproteins in which the flavin adenine dinucleotide prosthetic group is tightly bound to the enzyme protein. The optimal pH of two enzymes was 7 to 8 and the Michaelis-Menten constant was 3 to 5 x 10-4 m for each d-amino acid. Substrate specificity and thermal stability were observed to be somewhat different for the two enzymes. No inhibition of these enzymes was observed with metal chelating agents.