Aurora Brunner

In vivo modification of the energy charge in the liver cell

Summary The energy charge of the adenylate system increases in the liver cells of normal rats 30–120 minutes after the intra — peritoneal injection of adenosine (200 mg/Kg body weight). Simultaneously, in the liver there is an enhancement of glycogen biosynthesis and a diminution in fatty acid catabolism. These metabolic changes correspond to what could be expected from an increase in the energy charge. Thus, a regulatory role of the adenylate system under in vivo conditions is substantiated.

Cytoplasmic and nuclear inheritance of resistance to alkylguanidines and ethidium bromide in a petite-negative yeast

Abstract Mutants of the petite-negative yeast, Kluyveromyces lactis , resistant to the inhibitors of oxidative phosphorylation, decamethylenediguanidine and octylguanidine were isolated from medium containing ethidium bromide. All mutants were resistant to ethidium bromide; some mutants were resistant to both alkylguanidines, some to one, others to neither. Both nuclear and cytoplasmic inheritance of resistance to decamethylenediguanidine and ethidium bromide was demonstrated by tetrad analysis.

SOME CHARACTERISTICS OF THE D‐GLUCOSE‐6‐PHOSPHATE: CYCLOALDOLASE (NAD+ DEPENDENT) FROM Neurospora crassa *

The biosynthesis of myo-inositol? seems to be very similar in all species investigated, including yeast (Chen & Charalampous, 1963), mammals (Eisenberg & Bolden, 1963), higher plants (Kind1 & Hoffmann-Ostenhof, 1964), and Neurospora (Piiia & Tatum, 1967). A single enzyme, with an absolute requirement for nicotinamide adenine dinucleotide (NAD+) , catalyzes the formation of inositol-phosphate (inositol-P) from glucose-6-phosphate (glucose-6-P) . Eisenberg and Bolden (1965) and Chen and Charalampous (1966) have demonstrated for the mammal and yeast enzyme, respectively, that L-myoinositol1-phosphate is the enzymatically formed isomer. The present paper deals with some properties of the enzyme, isolated from Neurospora crassa, which is responsible for this reaction.

Utilization of adenosine as a tool in studies on the regulation of liver glycogen biosynthesis

An eightfold increase in the active form of liver glycogen synthetase activity was detected in normal rats 1 hr after the intraperitoneal injection of adenosine. Such an increase is the highest yet observed after the administration of a specific hormone or compound. However, no change was detected in the total quantity of a plus b forms of the enzyme present in the liver. Previous administration of actinomycin or cycloheximide prevented increases in the active form of glycogen synthetase activity ordinarily provoked by the injection of the nucleoside. The addition of adenosine to rat liver homogenates did not cause a change in the active or total glycogen synthetase activity as compared to the control. The effect of adenosine was not mediated by insulin sincein diabetic rats the injection of the nucleoside was also followed by an increase in the active form of liver glycogen synthetase activity. Variation in the levels of blood glucose and serum inorganic phosphate and changes in the levels of inorganic phosphate in liver cells were also observed after adenosine administration. The maximum changes preceded the increase in the active form of hepatic glycogen synthetase in vivo . It is postulated that in the liver the synthetase phosphatase responsible for the conversion of the b into the a form of glycogen synthetase is the important point of regulation of glycogen biosynthesis.

Protoplast fusion in a petite-negative yeast, Kluyveromyces lactis

SummaryThe technique of protoplast fusion has been applied to the problem of unstable diploidy in the yeast Kluyveromyces lactis. By protoplast fusion between heterothallic strains of like mating-type, sporulation-deficient hybrids can be obtained. Biochemical, cytological, and genetical characterisation of these hybrids suggests that the majority of fusion products are diploid. Sporulating hybrids can be constructed by protoplast fusion between homothallic strains. Tetrad analysis of these hybrids demonstrates conclusively the diploid nature of fusion products.

Behavior of cycloaldolase from Neurospora crassa towards substrate analogs and aldolase inhibitors.

Abstract Several characteristics of the d -glucose-6-phosphate; l -myo- inositol- l -phosphate cycloaldolase (NAD+-dependent) from Neurospora crassa were investigated. At a concentration of 5 mM, the following compounds inhibit the enzymatic reaction in decreasing order of effectiveness: d -sorbitol-6 -P , d -ribose-5 -P , d -fructose-6 -P , 6-phospho- d -gluconate, 5-keto- d -gluconate and d -galactose-6 -P . At similar concentrations d -glucosamine-6 -P , d -glucose-6-sulphate, myo-inosose, d -glucose and d -mannose-6-P fail to inhibit the activity. NADP+ does not substitute for NAD+ as coenzyme and NADPH is a very poor inhibitor compared to NADH. The reaction is not inhibited by pyridoxal-5′-P, trinitrobenzenesulphonate or NaBH4 which classically inhibit reactions where a Schiff base is formed between enzyme and substrate. The reaction was inhibited by high concentrations of EDTA.

Effect of (NH4)2 SO4 and glycerol on the preservation of the “NAD+-independent” activity of d-glucose-6-phosphate, l-myo-inositol-1-phosphate cycloaldolase from Neurospora crassa

Abstract Partially purified preparations of glucocycloaldolase from Neurospora crassa show an absolute requirement for NAD+. The experimental findings in this paper indicate that the conditions of isolation used determine whether or not glucocycloaldolase activity can be detected in the absence of added NAD+. The protective action of glycerol and (NH4)2SO4 on this so-called “NAD+-independent” activity was studied. Extraction of the enzyme in the presence of either 25% glycerol or 0.19 m (NH4)2SO4 preserved 80–90% of the “NAD+-independent” activity. Precipitation of the enzyme between 50–66.6% (NH4)2SO4 and dialysis of this fraction for 13 hr against Tris, Tris plus 25% glycerol or Tris plus 0.19 m (NH4)2SO4 preserved, respectively, 0, 30, and 70% of the “NAD+-independent” activity. Other salts containing either NH4+ or SO4−2 ions were not as effective as (NH4)2SO4 itself. Exogenously added NAD+ could be coprecipitated with glucocycloaldolase which was completely dependent upon added NAD+, to give rise to a preparation which was 41% “NAD-independent”. Studies of a purified glucocycloaldolase preparation, which gives only three bands on acrylamide gel electrophoresis, showed that addition of exogenous NAD+, followed by dialysis, gave 50% of the “NAD+-independent activity” provided the dialysis was effected against Tris containing 0.19 m (NH4)2SO4. Only 10% was recovered when Tris alone was used.

Correlation between resistance to ethidium bromide and changes in monovalent cation uptake in yeast

Abstract A mutant of Kluyveromyces lactis resistant to ethidium bromide was studied and found to have an impairment to transport the dye. As described for other mutants of this kind, the fluorescence changes of the dye that are observed when the cells transport it, were not observed in the mutant strain. Simultaneous to this difficulty to take up the mutagen, the cells showed a diminished ability to take up monovalent cations, as compared to the wild-type strains. The defect of the mutant strain does not seem to reside in the capacity to pump out protons, which also indicates that it has no alterations of the general energy conversion systems. This view is also supported by the fact that the growth yields are similar in both the mutant and the wild-type strains. Both ethidium and K + fail to stimulate respiration of the mutant yeast when present in the medium, as compared to the wild-type strains. The mutant strain shows a normal cation content, which indicates that the impairment to take up monovalent cations, although much decreased, may still be enough to maintain a normal content of cations within the cells. According to the investigation carried out, the mutant cells seem to be normal, expect for the fact that they are unable to transport both ethidium and K + from the medium. The data support the hypothesis that ethidium bromide and K + may be transported by the same system in yeast.

Ethidium bromide uptake and change of fluorescence by petite-negative yeast mutants resistant to this drug

The induction of cytoplasmic p-mutants by ethidium bromide(EB**) in the yeast Saccharomyces cerevisiae is a well known phenomenon [ 1 ] . These respiratory deficient mutants do not grow on nonfermentable substrates since their mitochondria are characterized by their abnormal content of respiratory enzymes and altered mitochondrial DNA [2] . The characterization and study of both nuclear and cytoplasmic yeast mutants resistant to EB is essential for the understanding of the mechanisms involved in the induction of respiratory deficient ‘petite’ mutants in the so-called petite-positive yeasts [3], by this drug whenever non-fermentable substrates are used as carbon sources in the growing media [l] . One cytoplasmic and two different classes of nuclear mutants resistant to EB have been isolated and genetically characterized from the petite-negative yeast Kluyveromyces lactis [4,5] in our laboratory. The uptake and change of fluorescence (P*) of EB using whole cells was studied in wild type and these mutant strains with the purpose of elucidating the mechanism of actions of this drug. R was found that the degree of uptake of EB is greatly dependent on the nature of the substrate. Significant differences were observed between the wild type and the mutant in their capacity to bind this drug.

The regulation of myo-inositol-1-phosphate-synthase activity from Neurospora crassa by pyrophosphate and some cations

The effect of several cations on the inhibition by PPi of the enzyme myo-inositol-1-phosphate synthase (1L-myo-inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4) from Neurospora crassa was studied. The study wastol biosynthesis can occur in the presence of an intracellular PPi concentration which exceeds the Ki for the enzyme by 350-fold. The inhibition of enzyme activity by PPi,at pH 7.7, was reversed, in decreasing order of effectiveness, by Mn-2, Fe-3