On the Constituents of a Chinese Rose Oil

Improvements were found in the inosine productivity of Brevibacterium ammoniagenes KY 13714, which is an adenine leaky and 6-mercaptoguanine resistant mutant. A highly productive mutant, KY 13761, was selected after the addition of 6-methyIthiopurine resistance and guanine requiring character to KY 13714 and after repeating single colony isolation.

Culture conditions for the practical production of inosine were investigated using KY 13761. It was found that the concentrations of phosphate, magnesium, and adenine were important. Carbon sources and natural nutrients also showed profound effects on inosine accumulation. Especially, effective was the feeding of inverted molasses and urea for production of inosine. Under optimal conditions, 31 mg of inosine per ml was accumulated after 42 hr cultivation in a 5 liter jar fermenter at 32°C. A growth-associated type of accumulation was confirmed in inosine production with KY 13761.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.     

Production of Nucleic Acid-Related Substances by Fermentative Processes. XXVIII. Accumulation of 5′ Inosinic Acid by a Manganese-Insensitive Mutant of Brevibacterium ammoniagenes

A manganese-insensitive mutant, KY 13105, of Brevibacterium ammoniagenes which accumulates considerable amounts of 5' inosinic acid (IMP) in the presence of 100 to 1,000 mug of Mn(2+) per liter was obtained from an IMP-producing mutant of a manganese-sensitive strain, KY 13102. The effects of Mn(2+) at 0 to 30 mug/liter on IMP accumulation by KY 13105 were similar to those by KY 13102. However, the accumulation of IMP by KY 13105 was not affected by 100 to 1,000 mug of Mn(2+) per liter, showing a clear difference from KY 13102. The accumulation of IMP by KY 13105 was always accompanied by cellular morphological changes irrespective of Mn(2+) concentration. In the presence of Mn(2+), factors which affect IMP accumulation by KY 13105 were examined. Most of the nutrients tested stimulated IMP accumulation at a relatively low concentration (2 g/liter). Iron, calcium, and zinc were found to be essential for IMP accumulation and were independent of Mn(2+). Biotin regulated the growth but not the accumulation of IMP. Under limited or surplus amounts of Mn(2+), the dynamics of IMP fermentation were followed. Under both conditions, the fermentations proceeded in a similar way. The morphological changes were found to be closely related to IMP accumulation.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Brevibacterium insectiphilium KY 3446 (Steinhous, Breed AHU 1401) was found to accumulate IMP from hypoxanthine and UMP from uracil, respectively. This strain is thus considered to present the fourth example in salvage-type fermentation, in addition to Micrococcus sodonensis, Arthrobacter citreus and Brevibacterium ammoniagenes reported previously.

IMP from adenine and UMP from cytosine were also produced by KY 3446, respectively. Further, the addition of inosine and adenosine instead of the bases also caused IMP accumulation.

This strain grew well on sucrose medium, and produced IMP and UMP in higher yields on sucrose than on glucose medium.

Excessive amounts of Mn²⁺ stimulated growth, but markedly inhibited IMP production. The optimal concentration of Mn²⁺ for IMP accumulation induced morphogenetic alterations from normal and small to abnormal and large cells.     

Nature of 6-methylpurine inhibition and characterization of two 6-methylpurine-resistant mutants of Neurospora crassa.

6-Methylpurine, an analog of adenine, inhibits the growth of Neurospora crassa. From kinetic studies it was found that 6-methylpurine is converted to its nucleotide form by adenine phosphoribosyltransferase (EC 2.4.2.7), and inhibits the de novo purine biosynthesis. Adenine relieves the growth inhibition caused by 6-methylpurine, whereas hypoxanthine is not very effective. Studies dealing with hypoxanthine utilization in the presence of 6-methylpurine indicated a severely reduced uptake of hypoxanthine and a general slowdown in its further metabolism. Two mutants (Mepr-3 and Mepr-10) which are resistant to 6-methylpurine were characterized. Studies of purine base uptake and the in vivo and in vitro conversion to nucleotides indicated that Mepr-10 may be an adenine phosphoribosyltransferase-defective mutant, whereas Mepr-3 may be a mutant with altered feedback response to 6-methylpurine. Both mutants showed a severely lowered hypoxanthine phosphoribosyltransferase activity, but because 6-methylpurine did not have any effect on the conversion of hypoxanthine to IMP in the wild type, it was concluded that 6-methylpurine resistance in these mutants cannot be due to lowered hypoxanthine phosphoribosyltransferase activity, but rather that the lowering of enzyme activity may be a secondary effect.     

Production of Nucleic Acid-Related Substances by Fermentation Processes

The effects of manganese ion (Mn²⁺) and adenine on the accumulation of 5′ inosinic acid (IMP) by Brevibacterium ammoniagenes KY 13102, were examined. Adenine regulated the accumulation of IMP in the presence of limiting amounts of Mn²⁺ and the accumulation of hypoxanthine (Hx) in the presence of excessive amounts of the ion. Manganese ion markedly affected IMP accumulations, cell growth and cellular morphology. These biological changes caused by Mn²⁺ are related to changes in the syntheses of macromolecules. The cells cultivated under limitation of Mn²⁺ showed abnormally elongated and irregular forms irrespective of adenine levels and had smaller nucleotide pools than those of the cells in the presence of excessive Mn²⁺. The Mn²⁺ limited cells showed ability to accumulate IMP directly in the cell suspension but the Mn²⁺ excessive cells did not accumulated IMP but Hx. These results indicated that adenine and Mn²⁺ affected the IMP accumulation independently each other and adenine acted as a feedback regulator on de novo synthesis of purine nucleotide and limitation of Mn²⁺ caused morphological changes, resulting in changes of permeability of the cells. The fatty acid contents of the Mn²⁺ limited cells were higher than those of the Mn²⁺ excessive cells and the ratio of unsaturated fatty acid to saturated one was higher in the former cells.     

Regulation of 5-phosphoribosyl 1-pyrophosphate and of hypoxanthine uptake and release in human erythrocytes by oxypurine cycling.

Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically.     

Pentatrichomonas hominis: purine salvage pathway

1. Pentatrichomonas hominis was found incapable of de novo synthesis of purines. 2. Pentatrichomonas hominis can salvage adenine, guanine, hypoxanthine, adenosine, guanosine and inosine, but not xanthine for the synthesis of nucleotides. 3. HPLC tracing of radiolabelled purines or purine nucleosides revealed that adenine, adenosine and hypoxanthine are incorporated into adenine nucleotides and IMP through a similar channel while guanine and guanosine are salvaged into guanine nucleotides via another route. There appears to be no direct interconversion between adenine and guanine nucleotides. Interconversion between AMP and IMP was observed. 4. Assays of purine salvage enzymes revealed that P. hominis possess adenosine kinase; adenosine, guanosine and inosine phosphotransferases; adenosine, guanosine and inosine phosphorylases and AMP deaminase.     

Accumulation of 6-Azauridine by Mutants of Brevibacterium ammoniagenes

From the inosine producing mutants of Brevibacterium ammoniagenes, KY 13761 was selected as a strain which produced 6-azauridine from 6-azauracil.

The conditions for the conversion were examined and the intermitent feeding of 6-azauracil was found effective for the accumulation.

In order to increase the accumulation, prototrophic revertants were induced from KY 13761 and KY 13021 was selected. By intermitent feeding of 6-azauracil of a final concentration of 6 mg per ml, a maximal accumulation, 12.4 mg/ml, of 6-azauridine was obtained with KY 13021.     

Metabolism of 6-Mercaptopurine by Resistant Escherichia coli Cells

Coggin, Joseph H. (University of Chicago, Chicago, Ill.), Muriel Loosemore, and William R. Martin. Metabolism of 6-mercaptopurine by resistant Escherichia coli cells. J. Bacteriol. 92:446-454. 1966.-6-Mercaptopurine (MP) utilization as a source of purine in MP-sensitive and -resistant cultures of Escherichia coli was investigated. The label of MP-8-C(14) appeared in adenine and guanine of ribonucleic acid and deoxyribonucleic acid in sensitive and resistant cultures. Studies using MP-S(35) further demonstrated that the MP moiety was degraded, as shown by a rapid decrease in radioactivity from cells upon exposure to MP for 20 min. Enzymatic analysis showed that MP was converted to 6-mercaptopurine ribonucleotide (MPRP) by extracts derived from both sensitive and resistant cells. Resistant cell preparations, however, degraded MPRP to inosine monophosphate (IMP) rapidly when compared with analogue degradation by sensitive cells. Inosineguanosine-5'-phosphate pyrophosphorylase from resistant cells did not catalyze the synthesis of IMP from hypoxanthine when the cells were cultured in the presence of MP, but these enzyme preparations actively converted guanine to guanosine monophosphate (GMP). Pyrophosphorylase derived from resistant cells cultured in medium without MP catalyzed the conversion of hypoxanthine to IMP and also guanine to GMP. These observations suggest that inosine-guanosine-5'-phosphate pyrophosphorylase is composed of two distinct enzymes. The mode of resistance to MP in E. coli is related to an enhancement of the enzymatic degradation of MPRP to the pivotal purine intermediate, IMP.     

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Inhibition of Dictyostelium discoideum beta-glucosidase by purines.

beta-Glucosidase of Dictyostelium discoideum is inhibited by purines in the following order: adenine greater than adenosine greater than 6-methylaminopurine greater than hypoxanthine greater than inosine greater than purine greater than guanosine. Adenine inhibits activity by 50% at 1 to 2 mM. The kinetics are complex because the enzyme is stimulated by substrate and inhibited by glucose.     

Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.     

Studies on 5′-Nucleotidase-Lacking Mutants Derived from Bacillus subtilis

For the purpose of effective accumulation of 5′-MMP, mutants, whose 5′-IMP-dephosphorylating activities were lower than that of strain A-1 of B. subtilis capable of accumulating a small amount of 5′-IMP as well as inosine and hypoxanthine, were derived from inosine-producing strain 1145-2-83 and strain A-1.

As a result, several mutants different from one another in the level of 5′-IMP-dephos- phorylating activity were isolated. Any of them did not acquire high ability to accumulate 5′-IMP. The more the mutants lost 5′-IMP-dephosphorylating activity, the less they accumulated extracellular inosine. The loss of nucleotide-dephosphorylating activity in the adenine-requiring mutants resulted in a remarkable increase in the amount of adenine required. The accumulation of 5′-IMP was not repressed by the addition of adenine at the concentration enough to repress accumulation of inosine.     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.     

Antitumor and Antiviral Properties of Penicillic Acid

The effects of adenine and (or) guanosine concentration on the accumulation of inosine, xanthosine, adenosine and succino-adenosine were studied with various purine auxotrophs of Bacillus subtilis K strain. Genetical derepression of the common pathway enzymes resulted in increase in the accumulation of inosine, xanthosine and adenosine. Co-operative repression system of a common pathway enzyme, succino-AMP lyase with respect to adenine and guanosine, was confirmed under the condition of the accumulation test. From these and the relating other studies it was concluded that the synthesis of AMP was regulated mainly by the inhibition of PRPP amidotransferase by AMP and secondly by the repression of the common pathway enzymes by adenine and guanosine, that the synthesis of GMP was regulated mainly by the inhibition and repression of IMP dehydrogenase by guanine derivatives and that GMP was synthesized in preference to AMP at the branch point, IMP.     

Guanosine metabolism in Neurospora crassa

Two aspects of guanosine metabolism in Neurospora have been investigated. (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine. The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.