SH Enzyme Research Articles

An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.

BackgroundSoluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported.ResultsDerepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility.ConclusionsWe report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0226-4) contains supplementary material, which is available to authorized users.

Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.

Reduction of arsenate to arsenite in hepatic cytosol.

After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (P(i)) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a P(i)-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD(+) or NADP(+)), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2-3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80-100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80-90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper.

Role of Protein -SH Groups in Redox Homeostasis— The Erythrocyte as a Model System

The reactivities of the sulfhydryl groups of rat, turkey, human, and calf hemoglobin were studied together with the enzyme activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutaredoxin in lysed erythrocytes to evaluate their roles in regulating redox homeostasis. The results of -SH reactivity showed rate constants spanning four orders of magnitude (k2, calf, 6.67 M−1s−1; rat -SH fast reacting, 2.8 × 104M−1s−1). Enzyme activities of glucose-6-phosphate dehydrogenase ranged from 0.402 U/ml (calf) to 0.900 U/ml (rat), glutathione reductase from 0.162 U/ml (rat) to 0.381 U/ml (human), glutaredoxin from 0.778 U/ml (rat) to 2.28 U/ml (turkey), and glutathione peroxidase from 2.07 U/ml (human) to 27.3 U/ml (rat). Blood samples of the four species were also treated with 0.5–1.5 mMtert-butyl hydroperoxide (t-BOOH) or diamide, and levels of glutathione-derived species [GSH, GSSG, and glutathione–protein mixed disulfides (GS–SP)] were determined within 120 min and related to the corresponding protein -SH group (PSH) reactivities and enzyme repertoires. In all cases t-BOOH rapidly transformed GSH into GSSG by the action of glutathione peroxidase; GSSG was in turn transformed into GS–SP, according to the reaction GSSG + PSH → GS–SP + GSH, or reduced back to GSH by glutathione reductase. The GSSG reduction was more efficient in rat and human blood, due to the contribution of the fast-reacting -SH of hemoglobin, in the rat, and to the efficiency of the enzyme repertoire of human blood. Calf blood showed a relatively low capacity to restore normal values after oxidative stress, due to its low PSH reactivity and the weak contribution of its enzymes. Diamide treatment, which is known to react nonenzymatically with thiols, gave increased GS–SP levels in rat and turkey, but not in human and calf blood, as expected from the different corresponding PSH reactivities. Species with relatively high PSH reactivity and glucose 6-phosphate dehydrogenase activity, such as the rat, therefore had a higher antioxidant capacity than species (calf) in which these parameters were relatively low.

Comparison of antimelanoma effects of 4-S-cysteaminylphenol and its homologues.

4-S-Cysteaminylphenol (4-S-CAP), a phenolic thioether, has been evaluated for melanocytotoxicity. We have recently shown that dihydro-1,4-benzothiazine-6,7-dione (benzothiazine BQ) is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-SCAP. In this study we compared the antimelanoma effects of 4-SCAP and its two homologues, alpha-methyl-4-S-cysteaminylphenol (alpha-Me-4-SCAP) and 4-S-homocysteaminylphenol (4-S-Homo-CAP). Biochemical experiments showed that upon tyrosinase oxidation alpha-Me-S-CAP and 4-S-Homo-CAP also produced homologues of BQ which reacted rapidly with reduced glutathione (GSH) and also inhibited alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that 4-S-CAP and its two homologues were taken up into B16-F1 melanoma cells at comparable rates but that 4-S-Homo-CAP was least effective in GSH deprivation, which was reflected in the low cytotoxicity of this phenol, and that the cytotoxicity of the phenols was tyrosinase dependent, as proved by the negligible effects on B16-G4F cells which have a much lower tyrosinase activity. In vivo experiments showed that direct intratumoral administration of these phenols inhibited the subcutaneous growth of B16 melanoma, with 4-S-Homo-CAP being the least effective, and that indirect Intraperitoneal administration of 4-S-CAP inhibited melanoma growth much more effectively than the two homologues. These results indicate that 4-S-CAP is the most promising antimelanoma agent among the three phenols examined.

Dihydro-1,4-benzothiazine-6,7-dione, the ultimate toxic metabolite of 4- S-Cysteaminylphenol and 4- S-Cysteaminylcatechol

4- S-Cysteaminylphenol (4- S-CAP) and the corresponding catechol 4- S-cysteaminylcatechol (4- S-CAC) have been evaluated for melanocytotoxicity. It was shown recently that tyrosinase oxidation of these substrates produces a violet pigment, dihydro-1,4-benzothiazine-6,7-dione (BQ). In this study we examined whether BQ is the ultimate toxic metabolite produced in melanoma cells from 4-S- CAP 4-S- CAC . Biochemical experiments showed that (1) BQ was formed by autoxidation of 4- S-CAC as well as by tyrosinase oxidation of 4-S- CAP 4-S- CAC , (2) BQ reacted rapidly with thiols such as reduced glutathione (GSH), and (3) BQ inhibited the activity of alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that (1) the cytotoxicity of 4- S-CAC was mostly prevented by catalase and superoxide dismutase, (2) BQ was highly cytotoxic to B16 melanoma cells ( ic 50 being 3.9 μM as compared with 507 μM for 4- S-CAP), (3) BQ was metabolized rapidly to a GSH adduct in melanoma cells, and (4) the same GSH adduct was also formed upon incubation of melanoma cells with 4- S-CAP, the reaction being tyrosinase dependent. In vivo experiments showed that intratumoral administration of BQ (0.5 μmol) inhibited the subcutaneous growth of B16 melanoma nearly as effectively as 4-S- CAP 4-S- CAC (20 μmol). These results indicate that BQ is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-S- CAP 4-S- CAC . BQ deprives melanoma cells of GSH and may inactivate SH enzymes essential for DNA synthesis and cell proliferation by covalent binding through their cysteine residues, thereby exerting melanocytotoxicity. Cytotoxicity of 4- S-CAC depends mostly on autoxidation producing BQ and active oxygens.

Chemical Reactivity of Oxidation Products of the Dithiolanylidenemalonate Fungicide, Isoprothiolane.

Diisopropyl dithiolanylidenemalonate (isoprothiolane) was oxidized with m-chloroperbenzoic acid to give a monosulfoxide, disulfoxide, and disulfone. The monosulfoxide was subjected to addition reactions with such nucleophiles as methanol, thiols, and amines at the thioacetal carbon to open the dithiolane ring, affording a thiosulfinate, sulfinate and disulfide. The presence of a small amount of sodium carbonate accelerated the reactions and, moreover, reformed isoprothiolane from the ring-opened addition products. The further oxidation products were transformed into dithianes by reacting with nucleophiles. Isoprothiolane monosulfoxide inhibited alcohol dehydrogenase, an SH enzyme.

Purification and properties of purine nucleoside phosphorylase from Brevibacterium acetylicum ATCC 954.

Purine nucleoside phosphorylase of Brevibacterium acetylicum ATCC 954, which catalyzes the production of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a potent antiviral agent, from purine nucleoside and 1,2,4-triazole-3-carboxamide in a high yield, was purified 49-fold. This enzyme had a molecular weight of 31,000 and was a monomer. The isoelectric point of the enzyme was 4.7. The optimal temperature and pH of inosine phosphorolyzing reaction catalyzed by the enzyme was around 8.5 and 70 degrees C, respectively. The Michaelis constants for inosine, guanosine, and ribavirin were 1.43 mM, 2.44 mM and 2.08 mM, respectively, at 40 degrees C. This enzyme appeared to be a SH enzyme because it was inactivated by SH reagents, p-chloromercuribenzoate and N-ethylmaleimide, and HgCl2. In addition, this enzyme was completely inactivated by AgNO3 and was slightly inhibited by CuSO4. It showed nucleoside-phosphorolyzing activity toward inosine, 2'-deoxyinosine, 2',3'-dideoxyinosine, guanosine, 2'-deoxyguanosine, and xanthosine. However, adenosine and its derivatives could not be phosphorolyzed. This enzyme could not also phosphorolyze various 5'-mononucleotides. According to the amino terminal sequence analysis, the twenty residues from the amino terminal end of this enzyme were identified as follows: MTVNWNETRS-FLECKMQAKPE.

Biochemical characterization of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase in rat spleen microsomes.

Acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) ultrasonic disruption in the presence of 25% glycerol from rat spleen microsomes. About 26% of the enzymatic activity was recovered in the 225,000 X g supernatant by this treatment, although the specific activity was slightly decreased compared with the original microsomes. The solubilized enzyme was remarkably susceptible to various kinds of metal ions. Sulfhydryl reagents such as p-chloromercuribenzoate and N-ethyl-maleimide significantly inhibited the enzyme reaction, suggesting that the enzyme is an SH enzyme. Based on the sedimentation pattern in sucrose density centrifugation, the isoelectric point, the kinetic characteristics and the sensitivity to tryptic digestion of microsomes, it appears that acetyl-CoA:lyso-PAF acetyltransferase does not differ from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to 1-acyl-2-lyso-sn-glycero-3-phosphocholine.

A phospholipase C with a high specificity for platelet-activating factor in rabbit liver light mitochondria.

The light mitochondrial fraction from rabbit liver was found to catalyze the hydrolysis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the phospholipase C reaction to form 1-O-alkyl-2-acetyl-glycerol and phosphocholine. The highest specific phospholipase C activity occurred in the liver and kidney. A subcellular survey showed that the enzyme was of lysosomal origin. The enzyme was solubilized with 2% Triton X-100 from rabbit liver light mitochondria and purified ca. 600- to 700-fold with a 17% yield using procedures that included hydroxyapatite, Sepharose 4B and isoelectric focusing column chromatography followed by fast protein liquid chromatography. The enzyme consists of two forms having a pl of 4.7 and 5.8. Each form was purified to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme migrated to positions corresponding to apparent molecular weights of 33,000 and 75,000, respectively. The purified enzymes of pl 4.7 and 5.8 had pH optima of 8.2 and 8.5 and apparent Km values of 55.6 and 45.5 microM for PAF, respectively. Furthermore, their phospholipase C activity was significantly inhibited by the addition of 1 mM EDTA. EDTA-inactivated enzyme, however, recovered completely upon addition of Ca2+ to the original level. p-Chloromercuribenzoate markedly inhibited enzyme activity, suggesting that phospholipase C is a -SH enzyme. The physiological role of the enzyme should be evaluated, considering its specificity for a highly potent, biologically active ether-phospholipid.

The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress.

An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in the presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the 0 and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and 0 mM glucose is due to NADPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined.

Renal high-molecular-weight renin: unusual formation in the aged stroke-prone spontaneously hypertensive rats.

A high-molecular-weight renin (HMWR) was detected in the plasma (molecular weight 50,600) and renal cortical homogenate (molecular weight 57,000) of 25-week-old male stroke-prone spontaneously hypertensive rats (SHRSP), in contrast to the renin with a molecular weight of 38,000 in normotensive Wistar Kyoto rats (WKY) and 5-week-old SHRSP. However, renin granules contained only the renin with a molecular weight of 38,000, indicating that the renal HMWR is not a native form but is probably a renin/renin-binding protein complex. Such HMWR was not produced when the renal cortex was homogenized with an equal amount of renal cortex of WKY. Further, the HMWR of the aged SHRSP was converted into the 38,000-dalton renin, when incubated with the extract of renal cortex of WKY. Thus, the existence of a renal cortical substance that converts the renal HMWR into the 38,000-dalton renin was evidenced. This substance was fractionated with DEAE-cellulose and was characterized as a putative SH enzyme. We conclude that a deficiency in the HMWR-converting substance may be attributed to the unusual formation of HMWR in the aged SHRSP.

A novel tumor-associated molecular species of 5′-nucleotide phosphodiesterase

5′-Nucleotide phosphodiesterase (5′-NPDase) was partially purified from plasma membranes of murine lymphoma L5178Y. The enzyme was inactivated by N-ethylmaleimide and Zn 2+, but stabilized by dithiothreitol, suggesting that it is an SH enzyme. The enzyme, Km 1.54 mM, pI 5.8 and MW 23k, differs from liver 5′-NPDase in MW, Km and sensitivity to some inhibitors. On the contrary, 5′-NPDase, derived from normal mouse organs, is similar to the liver enzyme. The results suggest that tumor cells possess a novel molecular species of 5′-NPDase.

Solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes.

The enzyme alkylglycerol monooxygenase was solubilized with 2% of Triton X-100 and partially purified approximately to 83-fold with a 36% yield after a procedure which included 6-aminohexyl-Sepharose and DEAE-cellulose column chromatography, followed by a sucrose density gradient centrifugation. The molecular weight of the native form was estimated to be approximately 400000 as judged by Sepharose 6B column chromatography in the presence of Triton X-100. The enzyme had a pH optimum near 8.5 and a Km of 0.66 mM for 1-O-hexadecylglycerol. The microsomal alkylglycerol monooxygenase was stimulated by Triton X-100, but did not affect kinetically the initial velocity except to maintain it for a much longer period of time. The purified enzyme required absolutely both molecular oxygen and reduced pteridine as an electron donor. Furthermore, reduced glutathione and phospholipids were necessary for expression of full enzyme activity. N-Ethylmaleimide and p-chloromercuribenzoate significantly inhibited the enzyme activity, suggesting that alkylglycerol monooxygenase is a -SH enzyme.

Studies on biologically active thiadiazolopyrimidines. Part V. Relationship between the structures and cytotoxic activities of 1,3,4-thiadiazolo(3,2-a)pyrimidines.

1, 3, 4-Thiadiazole derivatives having a partial structure of 2-ethylsulfonyl-7-methyl-5H-1, 3, 4-thiadiazolo[3, 2-α]pyrimidin-5-one (TPSO2-2) were synthesized, and their chemical reactivities and biological activities were investigated. TPSO2-2 readily reacted with compounds and showed a high inhibitory effect against SH enzyme, but 2-acetylamino-5-ethylsulfonyl-1, 3, 4-thiadiazole (AEST), a TPSO2-2 analog without a pyrimidine structure, did not react with those compounds and showed a smaller inhibitory effect against the enzyme. Furthermore, TPSO2-2 showed a strong anti-yeast activity, while AEST did not. It is presumed that not only the electron-withdrawing sulfonyl group at the 2-position but also the pseudopurine skeleton appear to be responsible for revealing the biological and chemical activities of TPSO2-2.

Mode of RNA synthesis inhibition by 1,3,4-thiadiazolo(3,2-a)pyrimidines.

When DNA-dependent RNA polymerase from Ehrlich ascites tumor cells (E-cells) was directly incubated with 2-ethylsulfonyl-7-methyl-5H-1,3,4-thiadiazolo[3,2-a]pyrimidin-5-one (TPSO2-2) and dialyzed to remove the chemical, its transcriptive activity was suppressed. The inhibition of enzyme activity was not restored by the addition of substrates. TPSO2-2 alkylated alcohols in the presence of amines. An alkylthio derivative which had no repressing effect on E-cell propagation did not react with alcohols. TPSO2-2 showed a high inhibitory effect against “SH enzyme,” such as yeast alcohol dehydrogenase, but was scarcely inhibited by an alkylthio derivative. TPSO2-2 reacted with l-cysteine. It is presumed that the reactivity of the 2-position of 1,3,4-thiadiazolo[3,2-a]-pyrimidines is responsible for biological activity and that RNA polymerase is inactivated by the alkylation of the SH or OH group with TPSO2-2.

Noncovalent binding of 4-nitroquinoline-N-oxide to proteins.

Binding of 4NQO to various kinds of enzymes or proteins was studied. Each one of proteins was mixed with 4NQO in 0.4 mM NaHCO3 solution and eluted through Ultrogel AcA 22 column. Radioactivity of 14C-labeled 4NQO found in protein fraction was measured. 4NQO bound hardly to polyglutamic acid and polyaspertic acid, somewhat to serum albumin, insulin, trypsin, RNA polymerase and DNA polymerase, and markedly to urease which is an SH enzyme. Lactate dehydrogenase, one of SH enzymes, aggregated with 4NQO. The binding of SH enzyme with the N-oxide would be attributable to a noncovalent binding such as 〉N-O---H-S-, because 4NQO-urease binding yield markedly decreased in the presence of sodium dodecyl sulfate or cysteine, and also 4NQO-bound urease released 4NQO by the addition of sodium dodecyl sulfate.

Soluble enzyme system for vitamin K-dependent carboxylation.

The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.

Suppression of long-day flowering by nitrogenous compounds in &lt;italic&gt;Lemna perpusilla&lt;/italic&gt; 6746

The long-day flowering of Lemna perpusilla 6746 on an SH inhibitor-containing medium was inhibited by the application of ammonium ion to the medium. Ammonium ion not only suppressed long-day flowering, but relieved the inhibition of vegetative growth caused by the inhibitors. Nitrite, casamino acids, glutamine and asparagine had a similar effect, suggesting that the inhibition of long-day flowering by ammonium ion is not a direct effect of the ion. Most amino acids, with the exception of glutamate and aspartate, also prevented long-day flowering, but their effects on vegetative growth varied. No qualitative differences in amino acid composition were observed among plants cultured on media containing nitrate, nitrite or NH44NO3as the sole nitrogen source. However, the amounts of free and total amino acids werehigher in plants fed with nitrite or NH4NO3 than in those fed with nitrate. This suggests that the inhibition of long-day flowering by ammonium and nitrite can be ascribed to increased nitrogen metabolism. Though decreased activity by SH inhibitors of nitrate reductase (SH enzyme) is assumed to result in long-day flowering by lowering the nitrogen metabolism, lowering the nitrogen level in M medium did not bring about floral initiation in the absence of SH inhibitors.

Purification and properties of an extracellular acid ribonuclease from Penicillium janthinellum

An extracellular acid ribonuclease produced by the mold Penicillium janthinellum has been purified 26-fold by a combination of selective heat treatment, ammonium sulphate precipitation and gel filtration on Sephadex G-200. The preparation gave a single band on electrophoresis on polyacrylamide gel at three different pH values. The enzyme has a pH optimum at 3.4. It is a heat-stable enzyme having no metal requirement but it is inhibited by divalent metal ions like Cu 2+, Zn 2+ and Hg 2+. It is a protein of mol. wt 30 000 and is not an -SH enzyme. It is a phosphotransferase catalyzing the hydrolysis of ribopolynucleotides to nucleoside-3′-monophosphates, via -2′,3′-cyclic mononucleotides. When -2′,3′-cyclic mononucleotides or homopolynucleotides are used as substrates the enzyme hydrolyses those containing uracil at a much faster rate than others.