G.L. Farkas

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.

Is the increase in ribonuclease level in isolated tobacco protoplasts due to osmotic stress

Abstract It has been shown in this experimental series that osmotic stress (plasmolysis induced by non-permeating plasmolytica), not mechanical or chemical injury, is the major factor which induces the large increase in the level of ribonuclease (RNAase) which occurs in isolated tobacco leaf protoplasts [1]. Protoplasts were isolated and incubated in media all of which contained the same standard ingredients but different amounts of mannitol. The RNAase activities measured in protoplasts after incubation for 48 h increased with the concentration of mannitol up to 0.7 M. Cycloheximide at a concentration of 1 μg/ml completely inhibited the increase in RNAase activity induced by hypertonic mannitol levels. Addition to the incubation mixture of 10−5 M kinetin or 10−5 M abscisic acid decreased or increased, respectively, the RNAase level that developed in the protoplasts during incubation for 48 h in hypertonic media. In leaf disks, floated on different osmotica with various permeability properties, an increase in RNAase level was detected only if the osmoticum induced plasmolysis. Ethylene glycol, a rapidly permeating osmoticum, did not increase the RNAase activity at all. In protoplasts incubated in the presence of 0.7 M mannitol large amounts of proline accumulated, as compared to the low proline level found after incubation in 0.4 M mannitol. This phenomenon is also indicative of osmotic stress associated with water loss.

Effect of "osmotic stress" on protein and nucleic acid synthesis in isolated tobacco protoplasts

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15–18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of 32P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.

The level of a relatively purine-specific ribonuclease increases in virus-infected hypersensitive or mechanically injured tobacco leaves

Abstract The increase in nuclease activity in ‘Xanthi’ tobacco leaves infected with tobacco mosaic virus (TMV) was found to be due mainly, if not entirely, to an increase in the level of a relatively purine (guanine) specific endoribonuclease. The level of the same nuclease increased in the leaf tissues upon mechanical injury. The level of other nucleases contained by the tobacco leaf remained unaltered both upon virus infection and mechanical injury. No correlation between virus multiplication per se and the increase in the level of the relatively purine specific ribonuclease was found.

Redox modulation of glucose‐6‐P dehydrogenase in Anacystis nidulans and its ‘uncoupling’ by phage infection

We have reported [l] that phage infection induces drastic changes in the respiratory carbon metabolism of Anacystis nidulans, a photoautotrophic cyanobacterium: (i) Glucose-6-P dehydrogenase (G6PDH), the first enzyme of the oxidative hexose monophosphate (HMP) pathway, undergoes a transition into a hyperactive form; (ii) Simultaneously with the activation of G6PDH, the carbon flow via the HMP shunt increases. In the heterotrophic prokaryote Escherichia coli phage infection has been reported to decrease [2,3] or leave unaltered [4] the activity of the HMP shunt. We report here that the different responses of the autotrophic and heterotrophic prokaryotes to phage infection can be explained by the different regulatory properties of their G6PDHs. In the healthy cyanobacterial cell, G6PDH is maintained in a low activity form by a powerful reducing system. Phage infection interferes with the operation of this reducing system, leading to an oxidative transformation of the enzyme into a hyperactive form. No similar regulatory mechanism is known to work in E. coli and other heterotrophic bacteria [5].

Changes in the level of acid phosphatases in Avena leaves in response to cellular injury

Abstract In illuminated Avena mid-leaf blades, the activity of various phosphatases increased with time. Overall activity increased by about 30 per cent during an incubation period of 8 hr. The activity was resolved into five peaks by molecular sieve chromatography. One of the peaks was purified and proved to be homogeneous upon chromatography on DEAE cellulose and upon polyacrylamide gel electrophoresis. The enzyme was characterized as a non-specific acid phosphatase splitting a wide range of substrates. The phosphatase exhibited regular Michaelis kinetics and was competitively inhibited by inorganic phosphate. Each tested substrate was a competitive inhibitor of the other. The molecular weight of the enzyme was found to be around 26,000.

Polyribosomes in Protoplasts Isolated from Tobacco Leaves

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.

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.