A Castelli

Specificity of lipids and coenzyme Q in mitochondrial NADH and succin-oxidase of beef heart and S. cerevisiae

Abstract The organic structural specificity of coenzyme Q in NADH- and succin-oxidase has been studied with mitochondria from the yeast, S. cerevisiae , and compared with the specificity for mitochondrial enzyme systems from beef heart. The specificity of lipids in CoQ-enzyme systems has also been studied. The origin and concentration of micellar phospholipids used as carriers for the CoQ homologs profoundly affects the restoration of NADH-oxidase in pentane-extracted mitochondria both from yeast and beef heart. High concentrations of phospholipids induce a decrease of the NADH-oxidase activity which had been restored by the lower homologs of CoQ. The activity with CoQ 1 -CoQ 4 was higher than that with the natural CoQ 6 in yeast mitochondria, and was lower than that with the natural CoQ 10 in beef heart mitochondria, irrespective of the concentration of phospholipids. Mitochondrial phospholipids of yeast were more effective than soybean phospholipids in supporting restoration of NADH-oxidase by the higher homologs in pentane-extracted mitochondria of yeast. This difference was not evident for succin-oxidase from yeast and beef heart. Succin-oxidase from both sources was similarly restored by the homologs CoQ 2 -Q 10 independently of the phospholipid concentration. According to these data, there are two sites for CoQ 6 in yeast mitochondria as there are two sites for CoQ 10 in beef heart mitochondria. The nature of the site in NADH-oxidase of yeast and of beef heart is different.

Effect of pH and CO2 concentration changes on lipids and fatty acids of Saccharomyces cerevisiae

SummaryThe lipid composition of S. cerevisiae cells was studied in different conditions of pH and of HCO3- and CO2 concentration.Increasing the concentration of CO2 in the broth determines an increase of total lipids, of total fatty acids, and of the relative amount of unsaturated fatty acids.Changing the concentration of bicarbonates at constant pCO2 does not affect the lipid composition of the cells. Also pH changes do not have significant effects.

Effect of hydroxy analogs of coenzyme Q on DPNH- and succin-oxidase activities of yeast mitochondria

Abstract 2,3-Dimethoxy-5-hydroxy-6-phytyl-and-6-farnesyl-1,4-benzoquinones (HPB and HFB) inhibit DPNH- and succin-oxidases of intact mitochondria from yeast. CoQ2 reversed the inhibition in DPNH-oxidase, but there was little or no reversal in succin-oxidase. CoQ6, the dominant CoQ of yeast, showed the same relative reversals as CoQ2. For CoQ6-deficient DPNH-oxidase, supplementation with: (a) HPB; (b) HFB; (c) HPB and CoQ6; (d) HPB and CoQ10 showed unexpected increases in activity indicating coenzymatic activity for HPB and HFB; the inhibitory effects of HPB and HFB were apparent with supplementation with CoQ2. HPB inhibited the CoQ6-deficient succin-oxidase, and also in the presence of CoQ2, CoQ6 or CoQ10.

Characterization of alkaline phosphatase inactivation by ascorbic acid

Ascorbic acid, isoascorbic acid and dehydroascorbic acid inhibit bovine kidney alkaline phosphatase activity. Ascorbic acid free radicals seem not to be involved. Dialysis does not make the inactivation reversible. A competitive mechanism can be inferred from experiments with phosphate and substrates, which block the activity decay. The influence of temperature, pH, other inhibitors and tertiary structure modifications on the inactivation process is also investigated.

Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.

No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.

Asynchronous development of different functions in mitochondria of saccharomyces cerevisiae

Yeasts have been considered very suitable organisms for the study of mitochondrial biogenesis. Irrespective of the genetic origin of the mitochondrial proteins [ 1,2] , little is known about how the mitochondrial membranes and their lipoprotein components are assembled together. Anaerobic yeasts have been shown to lack mitochondiia [3] ; some mitochondrial activities are nevertheless present [4] ; and the presence of mitochondrial membranes in these yeasts is determined by the nature of the growth medium [5], When anaerobically grown yeasts are allowed to grow in aerobiosis, mitochondria are formed [6], and there is parallel synthesis of the cytochromes [3]. The recognition by Green and his coworkers that membranes are made up of repeating units, containing fmed and detachable sectors [7], and the finding that the known functional activities of the mitochondrion are located within the sectors of the repeating units of the two membranes [8], open the way to a more precise study by biochemical means of the order by which the various mitochondrial components are synthesized and assembled. The present study deals with the development of some mitochondrial activities in yeast under controlled experimental conditions in which mitochondria pass from a repressed to an active stage. The results of this investigation demonstrate that membrane biosynthesis is a stepwise process in which the different components are independently formed, and that the assembly of these components precedes the emergence of integrated processes. Saccharomyces cerevisiae, strain ATCC 7754, was grown for 16 hr in a medium similar to that described by Klein [9] but containing 5% glucose, and then inoculated into a fermentor containing a rich me-

Effect of phospholipids from Saccharomyces cerevisiae at different stages of development on restoration of succinooxidase activity in lipid-depleted mitochondria

Phospholipids extracted fromSaccharomyces cerevisiae at different stages of development after glucose repression contain three major fatty acids: palmitic, palmitoleic and oleic. The ratio palmitic: palmitoleic strongly decreases beginning at the 6th hour of growth.To test the effect of fatty acid composition and in particular of unsaturation on succinoxidase activity, all these phospholipids, phospholipids from commercial yeast, and Asolectin were incubated with lipid-depleted yeast mitochondria. The amount of P bound was not much different for the various phospholipids; succinoxidase activity was restored best by Asolectin; the least effective reactivation was given by phospholipids from yeast at the middle stages of growth. There are not great differences between the various phospholipids and there is no correlation with unsaturation. If we compare the pattern of appearance of respiration during morphogenesis of yeast mitochondria with the pattern of the capability of the phospholipids from cells at different stages of mitochondrial morphogenesis to restore activity of lipid-depleted yeast mitochondria, we find no correlation. The results of this investigation are consistent with the idea that changes in phospholipids and changes in enzyme activities are not linked by a causal relation.

Biochemical properties of alkaline phosphatase from endometrial cancer cells

The occurrence of alkaline phosphatase (AP) activity was examined in several human endometrial adenocarcinomas. Catalytic activities were detectable only in 10 out of 15 tumors, with no apparent correlation between elevated AP and histological type. The apparent molecular weight of the enzyme after partial purification was about 140,000 daltons. Kinetic activity, thermodynamic properties and the pH dependence of the activity were in the ranges reported for other subforms. Several other physicochemical properties were also investigated and compared with those displayed by enzymes obtained from normal human tissues. The inhibition studies show that the enzyme shares several properties with the placental form, particularly in resistance to zinc chloride and EDTA action. On the other hand, in sensitivity to uncompetitive inhibitors and to urea and ascorbic acid, it is closer to other non-Regan heat-sensitive forms. The results support the view that a polymorphism in the expression of AP in neoplastic tissues can occur. A wider spectrum of physicochemical properties is clearly needed to define better the characteristics of oncodevelopmental enzymes.

Studies on the selective chemical inhibition by urea of alkaline phosphatase isoenzymes in the reaction course

Urea inhibits the activity of alkaline phosphatase during the reaction course. The inactivation is progressively stronger for the placental, intestinal and renal subforms. Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin and enzyme concentration on the inactivation mechanism is evaluated. In all experimental conditions the process follows pseudofirst-order kinetics and the inactivation profiles are distinct and typical for each enzymatic subform. With a simple graphical analysis, a single inactivation curve in controlled experimental conditions, allows the identification of each isoenzyme from the slope and the calculation of the respective fractional amount from the intercept of the time-activity plot.

Ascorbic acid and alkaline phosphatase activity

Ascorbic acid is found strikingly to decrease the activity of bovine kidney alkaline phosphatase in vitro. The inhibition of alkaline phosphatase is a function of ascorbic acid concentration and is time and temperature dependent. The presence of the substrate protects the enzyme against the inhibitory action of the vitamin.