R.J. Suhadolnik

Multiple forms of calf serum adenosine deaminase

Abstract Adenosine deaminase has been purified from calf serum by ammonium sulfate fractionation and DEAE cellulose column chromatography. This purified serum deaminase is ultracentrifugally homogeneous with an S 20 value of 3.9. This value contrasts with the S 20 value of 2.15 for calf intestine adenosine deaminase. Electrophoresis of the purified serum adenosine deaminase results in the separation of the protein into two major and two minor protein bands. Assay of the enzyme directly on the electrophoresis strips shows that all four protein components have adenosine deaminase activity. Comparison of the electrophoretic properties of calf serum deaminase with calf intestine deaminase reveals that the two major deaminase bands from the serum have the same electrophoretic migrations as the two deaminase bands from the purified intestine enzyme. The largest amount of protein is associated with the fastest protein band of the serum deaminase and the slower component of the intestine deaminase. Heating the calf serum enzyme to 60 ° for short periods of time converts the slow-moving deaminase band to the fast deaminase band. The K m , V max , energy of activation, pH optimum, and substrate or inhibitor specificity were measured using the purified serum adenosine deaminase. These values were compared to those obtained for the intestine deaminase and are reported here.

Toyocamycin: Phosphorylation and incorporation into RNA and DNA and the biochemical properties of the triphosphate

Abstract 1. Studies have been made on the biochemical properties of toyocamycin in Ehrlich ascites tumor cells and toyocamycin 5′-triphosphate as a phosphate donor. Toyocamycin, the naturally-occurring 7-cyano-7-deazaadenosine, is phosphorylated to the 5′-mono-, 5′-di-and 5′-triphosphates by Ehrlich ascites tumor cells. The three nucleotides were isolated in yields of 3.7, 1.2 and 0.43 %, respectively. The structures of the three nucleotides were established by chemical and enzymatic analyses and were similar to chemically synthesized mono-, di- and triphosphates. 2. Toyocamycin is incorporated into RNA and DNA. Toyocamycin in RNA was located in the internal and terminal positions. One radioactive spot was observed on a paper chromatogram after enzymatic hydrolysis of DNA. This suggests that toyocamycin is reduced to 2′-deoxytoyocamycin. 3. Toyocamycin triphosphate is 29 % as efficient as ATP as a phosphate donor with ATP: phosphoglycerate transphosphorylase. Toyocamycin triphosphate is not a phosphate donor with luciferase. The observed reactivities indicate a high degree of specificity by these enzymes for the adenine moiety of ATP. In contrast, 2′-dATP serves as a phosphate donor for both enzymes.

Incorporation of cordycepin (3′-deoxyadenosine) into ribonucleic acid and deoxyribonucleic acid of human tumor cells

Abstract Cordycepin (3′-deoxyadenosine), a nucleoside analog, is inhibitory to growing human tumor cells in culture. When these tumor cells were incubated with [3H]-cordycepin, the acid-soluble pool, the RNA and the DNA were radioactive. The RNA and DNA were hydrolyzed. The radioactive cordycepin was then isolated and crystallized to constant specific activities from the terminal and internal positions of the RNA. The cordycepin was also isolated and crystallized to constant specific activity from the DNA. In addition, the [3H]cordycepin, isolated after hydrolysis of the RNA (internal position) and the DNA, was further hydrolyzed to adenine and 3-deoxyribose. The 3-deoxyribose from the RNA (internal position) and the DNA was radioactive. No radioactivity was present in the 2-deoxyribose from the 2′-deoxyadenosine in the DNA. These results provide evidence that cordycepin is incorporated into the internal and terminal positions of RNA and the DNA and that 3-deoxyribose is not converted to 2-deoxyribose in H.Ep. No. 1 cells. The isolation of radioactive 3-deoxyribose from the cordycepin located in the internal position of the RNA indicates that a 2′,5′-phosphodiester bond is formed. The location (terminal or internal position) of the cordycepin in the DNA is not known.

INHIBITION OF HUMAN TUMOR CELLS BY CORDYCEPIN.

Abstract 1. Cordycepin inhibits the growth of human tumor cells (H.Ep. No. 1) grown in culture. The growth inhibition is cytostatic rather than cytocidal. 2. Adenosine competitively prevents inhibition but does not reverse it once it has occurred. This is in accord with the hypothesis that adenosine competes with cordycepin for phosphorylation to the active nucleotide level. 3. Exposure of human tumor cells (H.Ep. No. 1) to an inhibitory concentration of cordycepin does not markedly affect the protein, ribonucleic acid or deoxyribonucleic acid content. 4. Exposure to cordycepin results in a 2- to 4-fold depression of the incorporation of [8- 14 C]adenosine into ribonucleic acid and deoxyribonucleic acid. This is not in accord with the suggestion that the sensitive site for growth inhibition is early in the pathway of purine biosynthesis.

In vivo and enzymatic conversion of toyocamycin to sangivamycin by Streptomyces rimosus

Abstract The pyrrolopyrimidine nucleosides, toyocamycin, sangivamycin, and tubercidin are isolated from the culture filtrates of 14 species of the Streptomyces. Although earlier experiments showed that the biosynthesis of the pyrrolopyrimidine nucleosides require GTP as the common precursor, there was no experimental evidence to demonstrate the interconversion of these naturally occurring nucleoside analogs. The data presented here describe two types of experiments to prove that toyocamycin is the precursor for sangivamycin. First, in vivo experiments show that radioactive toyocamycin is converted to sangivamycin. Second, the enzyme, toyocamycin nitrile hydrolase, that catalyzes the conversion of toyocamycin to sangivamycin has been isolated and partially purified from the soluble fraction of S. rimosus. The nitrile hydrolase is not present in cell-free extracts of the Streptomyces that synthesize tubercidin or toyocamycin. Activity can be assayed by measuring the formation of radioactive sangivamycin from toyocamycin. The enzyme has been purified 24-fold with an over-all yield of 5%. The pH optimum is 6.5 and the K m is 0.5 m m . Most nitriles tested are competitive inhibitors but they are not substrates. The activity of the hydrolase is limited to the conversion of the nitrile group to the carboxamide group. Hydrolase activity is observed in cell-frre estracts of S. rimosus before toyocamycin production begins. The in vivo and in vitro studies demonstrate that toyocamycin is not a precursor for tubercidin. The experimental evidence strongly suggests that there must be a branch point in the biosynthesis of the pyrrolopyrimidine nucleoside antibiotics.

Nucleoside antibiotics: III. Isolation, structural elucidation and biological properties of 3′-acetamido-3′-deoxyadenosine from Helminthosporium sp. 215

Abstract Two additional nucleosides have been isolated from the culture filtrates of Helminthosporium sp. 215. They are 3′-acetamido-3′-deoxyadenosine and adenosine. The 3′-amino-3′-deoxyadenosine, isolated from the culture filtrates of Helminthosporium, was converted to 3′-acetamido-3′-deoxyadenosine by treatment with acetic anhydride. The chemical, physical properties and stereochemistry of the synthetic and naturally occurring 3′-acetamido-3′-deoxyadenosine were identical. The mass spectra as reported for 3′-acetamido-3′-deoxyadenosine and 3′-amino-3′-deoxyadenosine are very similar. The acetyl substituent on the 3′-amino-3′-deoxyadenosine position results in a somewhat different fragmentation pattern for 3′-acetamido-3′-deoxyadenosine when compared to 3′-deoxyadenosine. The mass spectrum of 3′-acetamido-3′-deoxyadenosine provides additional evidence for the structure of this nucleoside. The biochemical properties of 3′-amino-3′-deoxyadenosine and 3′-acetamido-3′-deoxyadenosine are markedly different. 3′-Acetamido-3′-deoxyadenosine was not inhibitory against Ehrlich-Lettre tumor cells nor bacteria while 3′-amino-3′-deoxyadenosine was toxic to ascitic adenocarcinoma. 3′-Acetamido-3′-deoxyadenosine was not toxic to mice, whereas 3′-amino-3′-deoxyadenosine was toxic to mice.

Nucleoside antibiotics IV. Metabolic fate of adenosine and cordycepin by Cordyceps militaris during cordycepin biosynthesis

Abstract The uptake and metabolism of uniformly 14C-labeled adenosine and the effect of cordycepin (3′-deoxyadenosine) and its 5′-phosphates on Cordyceps militaris have been studied. The uptake of [14C]adenosine by C. militaris was more rapid than the subsequent in vivo phosphorylation, conversion to cordycepin and incorporation into RNA. The distribution of 14C in cordycepin, the acid-soluble pool and RNA of cordycepin-producing cultures of C. militaris 24 h after the administration of [14C]adenosine was 24, 18 and 15%, respectively. Radioactive adenine, adenosine, AMP, ADP and ATP were isolated from the acid-soluble pool of C. militaris. A very small amount of uniformly 14C-labeled cordycepin was found in the acid-soluble pool. The purine nucleotides, AMP, GMP, IMP and XMP were isolated from the hydrolyzed RNA. Cell-free extracts of C. militaris rapidly hydrolyzed the 5′-mono- and 5′-triphos phates of cordycepin. Cordycepin and cordycepin 5′-monophosphate were not deaminated by adenosine aminohydrolase or AMP aminohydrolase. The 40 000 × g supernatant from C. militaris phosphorylated adenosine, 2′-deoxyadenosine, the pyrrolopyrimidine nucleoside antibiotics and psicofuranine but not cordycepin, uridine or cytidine. Ribosephosphate pyrophosphokinase from C. militaris was strongly inhibited by cordycepin 5′-triphosphate. Cordycepin 5′-monophosphate inhibited 5-phosphoribosyl-1-pyrophosphate amidotransferase. These studies in vitro suggest that these same enzymes that are inhibited by cordycepin 5′-mono- and 5′-triphosphates in Bacillus subtilis, pigeon liver and Ehrlich ascites tumor cells are also inhibited in C. militaris. Uniformly 3H-labeled cordycepin was not taken up by C. militaris. A 3′-ribonucleotide reductase, similar to the 2′-ribonucleotide reductase of Lactobacillus leichmannii and Escherichia coli, could not be demonstrated with cell-free extracts of C. militaris with adenosine, AMP, ADP or ATP as substrates. 3-Deoxyribose 5-phosphate was not a substrate for the biosynthesis of cordycepin in vitro.

Nucleoside antibiotics: V. The biosynthesis and interconversion of 3′-amino-3′-deoxyadenosine and 3′-acetamido-3′-deoxyadenosine by Helminthosporium sp. 215

Abstract Three nucleosides have been isolated from the culture filtrates of Helminthosporium sp. 215. They are adenosine, 3′-amino-3′-deoxyadenosine and 3′-acetamido-3′-deoxyadenosine. 3′-Acetamido-3′-deoxyadenosine appears in the culture filtrates first. At the end of nucleoside production, the molar ratio of 3′-amino-3′-deoxyadenosine: 3′-acetamido-3′-deoxyadenosine is 2:1, respectively. Uniformly 14C-labeled adenosine was incorporated into 3′-amino-3′-deoxyadenosine and 3′-acetamido-3′-deoxyadenosine without cleavage of the N- riboside bond. Uniformly 14C-labeled 3′-deoxyadenosine (cordycepin) is not a precursor for these nucleoside analogs. [1-14C]Acetate is incorporated exclusively into the acetyl group of 3′-acetamido-3′-deoxyadenosine. The interconversion of the two aminonucleoside analogs was determined by adding either uniformly 14C-labeled 3′-amino-3′-deoxyadenosine or 3′-acetamido-3′-deoxyadenosine to the culture filtrates of Helminthosporium at the time of nucleoside production. Both nucleosides are interconverted. The conversion of 3′-amino-3′-deoxyadenosine to the acetamidonucleoside is the most rapid. The distribution of 14C in the adenine and sugar moieties of the aminonucleosides is the same before and after interconversion. Therefore, no cleavage of the N-riboside bond took place. It is not known if this interconversion occurred in the culture filtrates or within the mycelium.

The biosynthesis of the 6-deoxy-D-erythro-2,5-hexodiulose sugar of decoyinine

Abstract 1. 1. 6-Deoxy- D - erythro -2,5-hexodiulose, the glucoside of the naturally occurring nucleoside, decoyinine, arises directly from D -[I- 14 C]glucose or uniformly 14 C-labeled D -fructose. 2. 2. Additional proof for the structure of this hexodiulose has been provided by the isolation of the C-6′ of decoyinine as iodoform. 3. 3. Psicofuranine, labeled with 14 C in the adenine and at C-6 of the D -psicose, is directly converted to decoyinine by Streptomyces hygroscopicus . The ratio of the 14 C in the adenine to that in the hexodiulose of decoyinine was the same as the ratio of the 14 C in the adenine to that in the psicose of the labeled psicofuranine added to the growing cultures of S. hygroscopicus . In addition, all of the 14 C in the hexodiulose of decoyinine resided at C-6. These results show that psicofuranine and decoyinine are interconverted.

Pseudouridine: Biosynthesis by Streptoverticillicum ladakanus

Abstract Recent studies have shown that pseudouridine in tRNA is formed by rearrangement of UMP residues. In the studies reported, information was not provided to clarify if the rearrangement of UMP residues to pseudouridine 5′-monophosphate was intramolecular or intermolecular. Experiments were designed to determine if the ribose moiety of the [U- 14 C]UMP in the tRNA of Streptoverticillicum ladakanus was transferred by an intramolecular rearrangement of the ribose from N-1 of uracil to C-5 of uracil. This was accomplished by comparing the ratio of 14 C in the aglycone: ribose of the UMP and CMP with the aglycone: ribose of the pseudouridine 5′-monophosphate of the tRNA. Because of the large amounts of pseudouridine excreted into the culture medium by S. ladakanus , it was also of interest to determine if there were a biosynthetic relationship between the pseudouridine found in the tRNA and the pseudouridine found in the culture medium. The results are as follows: (1) 33 % of the 14 C in the UMP and CMP resides in the ribose, (2) 20 % of the 14 C in the pseudouridine from the tRNA resides in the ribose, and (3) there is no 14 C in the ribose of the pseudouridine isolated from the culture medium. The 20 % 14 C ribose in the pseudouridine 5′-monophosphate isolated from the tRNA is taken as evidence that the biosynthesis of pseudouridine in tRNA occurs by an intramolecular rearrangement of pseudouridine 5′-monophosphate residues. The lack of 14 C in the ribosyl moiety of the pseudouridine isolated from the culture medium indicates that the pseudouridine in the culture medium is not formed from the turnover of pseudouridine 5′-monophosphate in the tRNA. Apparently, two different pathways are operational for the biosynthesis of pseudouridine in S. ladakanus . The exogenously supplied [U- 14 C]uridine is hydrolyzed by S. ladakanus as evidenced by the change in the ratio of 14 C in the uracil:ribose of the uracil isolated from the UMP and the cytosine:ribose of the cytidine isolated from the tRNA. [U- 3 H]Pseudouridine is taken up by S. ladakanus . However, it is neither degraded nor phosphorylated nor incorporated into cellular RNA. The absence of pseudouridylate synthetase in S. ladakanus is not the explanation for the large quantities of pseudouridine excreted into the medium because there was no correlation between the presence of pseudouridine in the culture medium with the absence of pseudouridylate synthetase activity in the five strains of Streptomyces studied.