An 8.5-kDa ribonuclease from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

Protein p3, a ribonuclease we previously isolated from the archaebacterium Sulfolobus solfataricus [P. Fusi et al. (1993) Eur. J. Biochem. 211, 305-310], was subjected to complete amino acid sequencing. It consisted of 75 residues, with a calculated M(r) of 8582, a pI of 10.1, and had some degree of monomethylation at Lys-4 and Lys-6. p2, a previously sequenced, 62-residue ribonuclease from the same organism, had an identical sequence for 57 consecutive residues starting from the N-terminus. p2 and p3 also showed a striking similarity to five other proteins previously isolated from Sulfolobus strains and identified as DNA-binding proteins. However, the C-terminus, 10 residue region of p3 did not show any similarity to these proteins; in contrast, it was significantly similar to stretches in three eubacterial ribonucleases from Bacillus strains. No difference between p2 and p3 has so far been detected as regards their catalytic properties. Available data suggest that these molecules have a narrow substrate specificity and probably play specific roles in RNA processing.

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH.

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) suppression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxy-kinase, but not of cytoplasmic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.

Ribonucleases from the extreme thermophilic archaebacterium S. solfataricus.

A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7-7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNA(fMet)Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides.

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from yeast Saccharomyces cerevisiae.

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.

Occurrence of two phosphorylated forms of yeast fructose-1,6-bisphosphatase with different isoelectric points.

Yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) immunoprecipitated from glucose-derepressed wild-type cells and subjected to isoelectric focusing, appears as a unique peak, essentially homogeneous and devoid of incorporated phosphate. However, after cell incubation with glucose, two phosphorylated forms are detectable. The isoelectric point of one is higher and of the other is lower than that of the native form. In contrast, in the mutant ABYS1 which is deficient in several vacuolar proteinases (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343), only the more acidic phospho form appears after cell incubation with glucose. However, sequence data rule out the possibility that limited proteolysis is the event responsible for the appearance of the more basic form of the phosphoenzyme. Nevertheless, time courses of glucose-induced inactivation of fructose-1,6-bisphosphatase show that the enzyme undergoes a substantially slower inactivation in the ABYS1 mutant as compared to the wild-type. These findings point to a degradative mechanism involving, besides the well-known phosphorylation, an additional as yet unknown modification which probably sensitizes the enzyme to proteolytic attack; furthermore, the enzyme responsible for such a modification seems to require one or more of the vacuolar proteinases missing in the mutant for its maturation.

Glucose-induced degradation of yeast fructose-1,6-bisphosphatase requires additional triggering events besides protein phosphorylation.

Glucose addition to yeast cells stimulates a cAMP overshoot with concomitant activation of cAMP-dependent protein kinase, which in turn rapidly phosphorylates fructose-1,6-bisphosphatase. The phosphorylated enzyme subsequently undergoes a slow proteolytic breakdown. Also, it has been proposed that phosphorylation represents the mechanism that initiates proteolysis. Here we present experiments carried out on a yeast mutant defective in adenylate cyclase [(1982) Proc. Natl. Acad. Sci. USA 79, 2355-2359] in which extracellular cAMP triggers full enzyme phosphorylation but a scanty proteolysis, whereas glucose plus cAMP provoke both phosphorylation and complete proteolytic breakdown. Thus, besides a glucose-induced cAMP peak, which results in enzyme phosphorylation, other effects evoked by the sugar are indispensable for its proteolytic degradation.

Inactivation and degradation of yeast glucose-6-phosphate dehydrogenase selectively modified by pyridoxal-5-phosphate.

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.

Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay.

Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+. The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer. Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase. Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.

Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants.

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.

L- and D-alanine transport in brush border membrane vesicles from lepidopteran midgut: evidence for two transport systems.

In brush border membrane vesicles from the midgut of Philosamia cynthia larvae (Lepidoptera) the L- and D-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition of L-alanine uptake by D-alanine is competitive, whereas inhibition of D-alanine uptake by L-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when Kout greater than Kin and Kout = Kin. D-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.

Susceptibility to proteinases of yeast enzymes selectively modified by fatty acids.

To investigate a possible correlation between selective modification and degradation of enzymes, the susceptibility to intracellular yeast proteinases A and B of yeast enzymes treated with fatty acids was tested. Enzymes used were glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), which are sensitive to the denaturing modification caused by fatty acids, and alcohol dehydrogenase (EC 1.1.1.1) which is insensitive. Proteinases and substrate enzymes were all pure preparations. Without modification by fatty acids, at neutral pH, the three enzymes are remarkably resistant to degradation by both proteinases. Treatment with myristic or oleic acid definitely enhances the susceptibility to proteolysis of the sensitive glucose-6-phosphate dehydrogenase and 3-phosphoglycerate kinase, whereas it leaves negligible that of the insensitive alcohol dehydrogenase. The selective effect of fatty acids on the degradation is pH-dependent: with proteinase A it was lost at acidic pH. Since intracellular levels of free fatty acids near or even higher than 1 mM were actually measured in yeast cells, it is possible that free fatty acids, in some cellular conditions, affect yeast enzyme composition. However, the control of specific enzyme degradation in yeast is still an open question.

Effect of caffeine on glucose-induced inactivation of gluconeogenetic enzymes in Saccharomyces cerevisiae. A possible role of cyclic AMP.

The mechanism of catabolite inactivation of three gluconeogenetic enzymes, fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase, has been studied in the yeast Saccharomyces cerevisiae. The glucose-induced inactivation of the three enzymes is remarkably retarded by preincubation of the cells with different caffeine concentrations; however, a full conservation of activity has never been obtained, even at the highest drug concentration. Caffeine modifies the metabolic effects produced in the yeast cell by exposure to glucose. It reduces the consumption rate of glucose; changes the glycolytic intermediate pattern, giving rise to a crossover point at the level of the phosphofructokinase/fructose-bisphosphatase cycle; and increases the ATP level and the energy charge. Moreover, it substantially reduces the peak of intracellular cAMP content that immediately follows glucose entry; the magnitude of this effect is dependent on the drug concentration. The effect on the change of intracellular cAMP level appears, among all metabolic effects determined by caffeine, the only plausible one to explain the interference with catabolite inactivation of enzymes. Actually a strong negative correlation between residual activity of each of the three investigated enzymes and intracellular cAMP level has been demonstrated. The existence of a common mechanism of action of cAMP, as the mediating factor for catabolite inactivation of all three enzymes, is proposed.

Reactivation of yeast glucose-6-phosphate dehydrogenase denaturated by saturated fatty acids.

Activity of yeast glucose-6-phosphate dehydrogenase, inactivated by treatment with saturated fatty acids, can be partially restored by incubation in a medium of suitable ionic composition. The effectiveness of ions in the reactivation process is inversely related to their 'chaotropic' properties. Time-dependence of reactivation extent suggests a 2-step mechanism of enzyme inactivation and the existence of an intermediate form that aggregates through a 2nd-order reaction, producing irreversibly inactive enzyme.

Selective denaturation of several yeast enzymes by free fatty acids.

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.

Regulation of enzymes of ethanol metabolism in yeast (Rhodotorula gracilis).

The three enzymes of ethanol metabolism alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase in the obligate aerobic yeast Rhodotorula gracilis are repressed by glucose and induced by C2 metabolic fuels with a regulatory pattern indicating a correlation in the control mechanisms. To try an identification of the molecular signals involved in the transmission of the inducing stimulus, experiments were carried out by blocking with 2 mM pyrazole the ethanol acetaldehyde metabolic step. Results indicate that ethanol is not specifically required as a molecular signal for induction.