J. Udvardy

Osmotic shock triggers an increase in ribonuclease level in protoplasts isolated from tobacco leaves

Abstract In protoplasts isolated from tobacco leaves and incubated in 0.7 M mannitol for 24 h, the ribonuclease (RNAase) level increased 12- to 15-fold during incubation. Various inhibitors of protein synthesis prevented or reduced the increase in RNAase activity. 10−5 M kinetin also reduced the increase in RNAase level. The increase in “overall” nuclease activity was accounted for by the accumulation in the protoplasts of the major nucleolytic enzyme of the tobacco leaf, a relatively purine (guanine)-specific endoribonuclease. The dramatic increase in RNAase level is due to the prolonged exposure of the isolated protoplasts to a medium of high osmotic value.

Interaction between hysteretic regulation and redox modulation of glucose-6-phosphate dehydrogenase from Anacystic nidulans

The glucose‐6‐phosphate dehydrogenase (G6PDH) of cyanobacteria is a hysteretic enzyme which is also subject to redox modulation [FEBS Lett. 126 (1981) 85–88]. We have found that the hysteretic and redox properties of G6PDH exhibit specific interactions: (1) The hysteretic forms of G6PDH (‘hypoactive’ ⇌ ‘hyperactive’), obtained at pH 7.5 and 6.5, respectively, differ in their redox properties. The ‘hypoactive’ form is easily activated by oxidation whereas the ‘hyperactive’ form is easily deactivated by reduction. (2) At low G6P concentrations (1 mM) only the oxidized form of G6PDH has significant activity. An increase in G6P level diminishes the difference between the activity of oxidized and reduced G6PDH forms.

Virus infection affects the molecular properties and activity of glucose-6-P dehydrogenase in Anacystis nidulans, a Cyanobacterium. Novel aspect of metabolic control in a phage-infected cell.

Infection of a bacterial ceti with bacteriophage is well known to res& in a ~~arnat~~a~ly altered pattern of nucleic acid and protein synthesis (or breakdown) in the host cell, In contrast, it has been stressed that the respiration of the host is not affected by phage infection [l], except at the onset of lysis [2]. Study of enzyme levels in cell-free extracts f3jt and investigation of the path of carbon in vitro [4j also revealed little effect of phage infection on the activities of respiratory enzymes. Thus it has been asssumed that the pre-existing respiratory machinery of the host cells can supply energy and C-skeletons in a suitable form and in sufficient amount for the new synthetic processes initiated by bacteriophage attack. Ail the work leading to the above conclusions has been done on heterotrophic bacteria, mainly Escherichiu coEi, infected with DNA phages. No information on the effect of phage infection on the respiratory enzymes or metabolism of autotrophic prokaryotes is available. The cyanobacteria are a major group of photosynthetic prokaryotes [S]. Some members of the group are attacked by specific bacteriophages (cyanophages) 161. Therefore the cya~obacterium~cyanophage system seemed promising for the investigation of the effect of phage infection on the respiratory metabolism of an autotrophic cell. We show here that the respiratory metabolism of Anacystis nidulans, a unicellular cyanobacterium, is drastically altered upon phage infection.

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and β-mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H2O2 led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H2O2. An oligomeric form of the enzyme (apparentMr∼440,000) was dissociated by GSH into a lower-Mr, more active enzyme form (Mr∼200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inKm and an increase inVmax values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.

The pyridine nucleotide-dependent D-glucose dehydrogenase of Nostoc sp. strain Mac, a cyanobacterium, is subject to thioredoxin modulation

A pyridine nucleotide‐dependent D‐glucose dehydrogenase (GDH) was isolated and purified about 1000‐fold from Nostoc sp. strain Mac. The activity of this preparation with NADP as cofactor was 2.8‐times that with NAD. This ratio did not change during purification. The enzyme both in crude extracts and after purification proved to be subject to redox modulation. Homologous and heterologous (Anacystics nidulans, Anabaena sp. strain PCC 7120, spinach) thioredoxins, in the presence of 0.5 mM DTT, deactivated the enzyme. The thioredoxin from Nostoc was active with heterologous enzymes: it activated the fructose‐1,6‐bisphosphatase of Anacystis nidulans and the NADP‐dependent malate dehydrogenase of spinach. The thioredoxin‐mediated reduction decreased the apparent V max value for D‐glucose by about 65% and that for NADP by about 51%. The apparent K m value for NADP increased upon reduction by about 10‐fold. The apparent K m value for D‐glucose was but slightly affected by the redox state of the enzyme.

Hysteretic behaviour of glucose-6-phosphate dehydrogenase of pea chloroplasts

Abstract Glucose-6-phosphate dehydrogenase (G6PDH) from pea chloroplasts has at least two interconvertible kinetic states which differ from one another in their catalytic activities (‘hyperactive’ and ‘hypoactive’ forms). Preincubation of chloroplast extracts with 10 mM glucose-6-phosphate (G6P) led to the accumulation of a ‘hyperactive’ G6PDH form which exhibited a burst of activity at the start of the assay; steady state was reached after a period of several minutes. Preincubation of the pea chloroplast extracts in the absence of G6P resulted in the formation of a ‘hypoactive’ enzyme from which exhibited a lag during the assay. Steady state was reached after several minutes. The enzyme activity in the steady state was the same for both forms. The length of the lag (τ) was inversely related to the concentration of G6DH and substrate concentration. These results show that the G6PDH of pea chloroplasts, like the enzyme of cyanobacteria, behaves as a hysteretic enzyme.

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

Abstract In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p -nitrophenyl phosphate or bis- p -nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe 3+ stimulated and Co 2+ and Ni 2+ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.

Redox regulation of glucose dehydrogenase from cells of the facultatively heterotrophic cyanobacterium Nostoc sp. strain MAC

Abstract A glucose dehydrogenase ( d -glucose:NAD(P)+ 1-oxidoreductase, EC 1.1.1.118(9)) was purified over 1100-fold, to near homogeneity, from Nostoc sp. strain MAC cells. The general enzymatic properties of the Nostoc enzyme were similar to those of the NAD(P)-dependent glucose dehydrogenase isolated earlier from facultatively heterotrophic cyanobacteria, heterotrophic bacteria and liver cells. In this work, we show that Nostoc glucose dehydrogenase can be modulated by various redox systems. Reducing agents such as dithiothreitol, cysteine and 2-mercaptoethanol decreased the activity of the enzyme. Reduced glutathione and ascorbic acid exerted only slight influence. The oxido-reductive modulation proved to be reversible. Treatment of the reduced glucose dehydrogenase with oxidants such as H2O2, Fe3+-EDTA and a Fe3+-chelate (Udvardy, J.; Borbely, Gy., Juhasz, A.; and Farkas, G.L. (1984) FEBS Lett. 172, 11–16) reversed the process of deactivation. The range of the reductive deactivation depended on pH, being the most effective in the alkaline range. Addition of 10 mM Mg2+ and Ca2+ in the cuvette to the reduced form of glucose dehydrogenase, however, reactivated the enzyme. These cations had little effect on the oxidized enzyme. Mg2+ and Ca2+ changed the kinetic parameters (Km and Vmax) of the reduced, but not of the oxidized form of the Nostoc glucose dehydrogenase. Redox modulation was not accompanied by any change in the apparent molecular mass (110–120 kDa).

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Abstract Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn 2+ and Fe 2+ were inhibitors for both by 20–80%; while Mg 2+ , Ca 2+ , Co 2+ , K + , Na + at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.