CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of gamma-phosphate from [gamma-32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [gamma-32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a gamma-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast system.     

Factors affecting killer activity of some yeast species occurring in rokpol cheese

Fourteen strains of Candida famata and 7 strains of C. sphaerica originating from blue-veined Rokpol cheese were studied for their ability to produce killer toxins against 3 strains of Yarrowia lipolytica selected as potential starter cultures for cheesemaking. All tested strains revealed killer activity in the presence of salt, at 14 degrees C and pH 4.6. Only one Y. lipolytica strain was resistant to C. famata and C. sphaerica killer toxins.     

Factors affecting the ethanol productivity of yeast in molasses

The ethanol production by a laboratory yeast strain, X2180-1B, was less than half that by an alcohol yeast, YOY655, in a molasses medium containing 30% sugars, although X2180-1B produced approximately the same amount of ethanol as YOY655 in a nutrition medium with the same sugar content. The weak productivity of X2180-1B in the molasses was ascribed to the limitation of sucrose hydrolysis in the molasses. The invertase activity of X2180-1B was 0.019 (mmol sucrose/min/mg protein) in the nutrition medium, but substantially zero in the molasses, while that of YOY655 was 1.75 in the nutrition medium and 1.15 even under the inhibitory conditions in molasses. External addition of invertase greatly enhanced the ethanol productivity of only X2180-1B. The inhibitory factors of invertase in molasses were heat-stable and dialyzable substances.     

Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca(2+) or Mg(2+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 degrees C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent K(m) = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K(m) = 0.3 mm). dCTP was both a substrate (apparent K(m) = 0.4 mm) and competitive inhibitor (apparent K(i) = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.     

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.     

Factors affecting lipid peroxidation in guinea-pig adrenal microsomes

Studies were carried out to examine the effects of and interactions between NADPH, Fe²⁺, Fe³⁺ and ascorbate on lipid peroxidation in guinea-pig adrenal microsomes. Fe²⁺, at levels between 10⁻⁶ and 10⁻³ M, produced concentration-dependent increases in lipid peroxidation in adrenal microsomes; Fe²⁺ had a far greater effect than Fe³⁺. In liver microsomes, by contrast, Fe²⁺ and Fe³⁺ had quantitatively similar effects on lipid peroxidation. NADPH alone had no effect on malonaldehyde production by adrenal microsomes. However, in the presence of low Fe²⁺ concentrations (10⁻⁶ M), NADPH stimulated malonaldehyde production; the stimulation was not demonstrable in microsomes which had been heat-treated to inactive microsomal enzymes. In the presence of high Fe²⁺ levels (10⁻³ M), NADPH produced a concentration-dependent inhibition of lipid peroxidation; the inhibition was fully demonstrable in heat-treated microsomes. In the presence of Fe³⁺ (10⁻⁶ to 10⁻³ M), NADPH had little effect on lipid peroxidation, suggesting that NADPH does not significantly promote the reduction of Fe³⁺ to Fe²⁺ in adrenal microsomes. Ascorbate alone increased malonaldehyde production by adrenal microsomes; maximum stimulation occurred at a concentration of 10⁻⁴ M. Ascorbate-induced lipid peroxidation was also inhibited by NADPH. Ascorbate (5 · 10⁻⁶ to 1 · 10⁻⁴ M) synergistically interacted with low levels (10⁻⁶ M) of Fe²⁺ to enhance malonaldehyde production by adrenal microsomes. The synergism was not demonstrable at high concentrations (10⁻³ M) of Fe²⁺ At all concentrations (10⁻⁶ to 10⁻³ M) of Fe³⁺ studied, ascorbate synergistically increased the production of malonaldehyde. The results indicate that interactions between various endogenous substances may be important in the control of adrenal microsomal lipid peroxidation and that there are differences in the regulation of adrenal and hepatic lipid peroxidation.     

Factors affecting invertase activity in soils

Summary The rate of reducing sugars released through invertase activity exhibited a buffer pH optimum of 5.0. Generally, the decline in invertase activity in its pH-profile near the optimal pH range was due to a reversible reaction that involved ionization or deionization of the functional groups in the active centre of the protein, but under highly acidic or alkaline conditions (pH<4 to >9) the reduced activity appears to be due to irreversible inactivation of the enzyme. The dependence of the reaction on the amount of enzyme present was linear up to 3 g of soil. By varying the substrate concentration, it was found that the reaction rate of this enzyme approached zero-order kinetics when 145mM of sucrose solution was added to soils. Application of three linear transformations of the Michaelis-Menten equation indicated that the apparent K_m constants varied among the soils studied, but the results obtained by the three plots were similar. By using the Lineweaver-Burk plot, the K_m values in five soils ranged from 16.3 to 42.1 (avg.=24.5) mM and V_max values ranged from 1.98 to 7.37 mg of reducing sugars released/g of soil/24 h. The optimum temperature for invertase activity in soils was observed at 50°C and denaturation of the enzyme began at 55°C. The activation energy (E_a) and enthalpy of activation (H^*) values for invertase activity, expressed in kJ/mole, ranged from 6.1 to 43.1 and 3.5 to 40.5, respectively. The Q₁₀ values for the invertase reaction in soils with a temperature range to 10 to 50°C ranged from 1.08 to 1.96. Under standerd conditions, the accumulation of reducing sugars was linear with time up to 48 h. Among the various pretreatments that affected invertase activity in soils, toluene, TCA, and PMA inhibited the enzyme by an average of 19, 54, and 11%, respectively. Steam-sterilization essentially destroyed soil invertase.     

Factors affecting the performance of crossflow filtration of yeast cell suspension

Factors affecting the performance of crossflow filtration were investigated with a thin-channel module and yeast cells. In crossflow filtration of Saccharomyces cerevisiae cells cultivated with YPD medium (Yeast extract, polypeptone, and dextrose) and suspended in saline, a steady state was attained within several minutes when the cell concentration was low and the circulation flow rate was high. The steady-state flux and the change in flux during the initial unsteady state were explained well by conventional filtration theory, with the amount of cake deposited and the mean specific resistance to the cake measured in a dead-end filtration apparatus used in calculation. When the circulation flow rate was lower than a critical value, a part of the channel of the crossflow filtration module was plugged with cell cake, and thus the steady-state flux was low. In crossflow filtration of suspensions of commercially available baker's yeast, the flux gradually decreased, and the flux after 8 h of filtration was lower than the value calculated by filtration theory. Fine particles contaminating the baker's yeast was responsible for the decrease. A similar phenomenon was responsible for the decrease. A similar phenomenon was observed in crossflow filtration of a broth of S. cerevisiae cells cultivated in molasses medium, which also contains such particles, had no effect of the permeation flux during crossflow filtration. (c) 1993 John Wiley & Sons, Inc.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

Factors affecting mycelial to yeast phase conversion and growth of the yeast phase ofHistoplasma capsulatum

A procedure for rapid induction of mycelial to yeast phase (M→Y) conversion ofHistoplasma capsulatum has been devised. Exposure of mycelial fragments to low oxidation-reduction (O/R) potentials (+5 to+65 mv) either aerobically (ascorbic acid treatment) or anaerobically (nitrogen atmosphere) for 18 to 24 hours at 37° C resulted in induction of the M→Y conversion process whether or not an organic sulfur source was available. However, aerobic conditions and a suitable organic sulfur source, such as cysteine, cystine or lanthionine were found essential for outgrowth and maintenance in the yeastlike phase.     

Factors affecting molecular packing in mixed lipid monolayers and bilayers

The nature of the molecular interactions and the factors determining molecular packing in mixed phospholipid/glyceride monolayers and bilayers were investigated by monolayer and nuclear magnetic resonance (NMR) techniques. Force-area curves were obtained at various temperatures for monolayers, at the air-water interface, of synthetic lecithins and a phosphatidyl ethanolamine mixed with di- and triglycerides in different molar ratios. The linewidths of peaks in the high resolution NMR spectra of lecithin/glyceride co-dispersions in excess water at different temperatures were used to obtain information about molecular mobilities.It was found that the molecular packing in mixed lipid monolayers and bilayers is determined by the following factors: (1) Whether lipid chains are above or below their melting point (T_C). (2) The difference between experimental temperature and T_C: the larger the difference, the smaller the effect of one component on the other. (3) The degree of similarity of the chains of the components; this influences the degree of cooperativity of chain motions and the degree of mixing of the components. (4) The nature, orientation, mutual interaction and degree of hydration of the polar groups.It is shown that mean molecular area does not always reflect the state of chain motions in lipid films, because of heterogeneity of motion and structure along the molecules. Cooperativity of motion may reduce steric requirements; other effects which are of particular importance for lecithins are interactions of zwitterions, and the influence of polar group hydration.     

Factors affecting the phospholipase activity of Candida species in vitro

The phospholipase activity of 41 isolates of oral Candida species was determined by a plate assay. Seventy nine per cent of the C. albicans isolates were phospholipase producers whereas none of the C. tropicalis, C. glabrata or C. parapsilosis isolates produced the enzyme. The degree of phospholipase activity (Pz value) of individual isolates was remarkably constant despite the large variation in activity among different isolates. Experiments with 10 phospholipase positive C. albicans isolates indicate that phospholipase production in vitro is limited to a narrow pH range (c. 3.6-4.7) and is suppressed by increasing concentrations of sucrose and galactose in the media (r = 0.9). Hence, candidal phospholipases seem to play a complex role in the aetiopathology of human candidoses.