Renato Bianchetti

HEXOSE MONOPHOSPHATE LEVEL AS A LIMITING FACTOR FOR RESPIRATION IN UNFERTILIZED SEA-URCHIN EGGS.

Abstract Quantitative changes of some glycolytic intermediaries have been investigated after fertilization of sea-urchin eggs. In the unfertilized eggs the concentration of all the sugar phosphates investigated was exceedingly low. Immediately after fertilization, the level of glucose 6-phosphate underwent a marked increase. The level of fructose diphosphate and triose phosphate, which are almost undetectable in the unfertilized egg, rose upon fertilization. Since the concentration of these esters is probably far below the point of saturation of the enzymes involved, breakdown of carbohydrates in the sea-urchin eggs may be limited by the low level of these esters. It is suggested that in the unfertilized egg there is a block of the pathway leading from glycogen to glucose 6-phosphate, and that release of this block takes place upon fertilization.

Initiation of protein synthesis in isolated mitochondria and chloroplasts

N5-Formyltetrahydrofolate, a competitive inhibitor of the formylation of the initiator Met-tRNAfMet in an in vitro assay, is a powerful inhibitor of amino acid incorporation in isolated Saccharomyces cerevisiae mitochondria and in Euglena gracilis chloroplasts. Thus, a large part of the incorporation is dependent upon new initiation acts. On the contrary, the rate of incorporation can be largely increased by addition of the specific formyl group donor, N10-formyltetrahydrofolate. Experiments are also reported strongly suggesting that the formylation of Met-tRNAfMet is an absolute requirement in order to initiate protein synthesis in chloroplasts, as has been shown in mitochondria.

Endogenous synthesis of formyl-methionine peptides in isolated mitochondria and chloroplasts

Abstract In the presence of a system for the incorporation of aminoacids, formyl group donor and puromycin, mitochondria and chloroplasts can initiate “in vitro” the synthesis of peptides with formyl-methionine at the N-terminus. This indicates that in these organelles endogenous messenger(s) programme polypeptide chains starting with formyl-methionine.

The mechanism of the repression by inorganic phosphate of phytase synthesis in the germinating wheat embryo.

It has been previously shown that phytase is repressed by Pi in isolated wheat embryos. The mechanism of the repression of phytase by Pi was investigated separately in the germ and in the scutellum of germinating wheat embryos. 1. 1. In the scutellum, the increase of phytase during germination is completely abolished by puromycin or by actinomycin. The supply of Pi specifically represses the synthesis of the enzyme. 2. 2. In the germ, puromycin also blocks the increase of phytase; actinomycin only delays it, while Pi has no effect on the synthesis of the enzyme. 3. 3. In the scutellum, the maximum value of the enzyme is reached at about the 30th h of germination. Experiments with actinomycin show that phytase synthesis is completely blocked only if actinomycin is supplied within the first 6 h of germination, while the inhibitor, when administered after the 14th h, is completely ineffective. Similar experiments show that Pi also has no effect on phytase synthesis if supplied after 6 h of germination. These results are interpreted as indicating that Pi is effective in repressing phytase synthesis only if supplied during a period coinciding with that of phytase messenger RNA synthesis. It is concluded that Pi acts at the transcription level.

Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase by the Plasmalemma Proton Pump in Acer pseudoplatanus L. Cultured Cells

Summary The involvement of the plasmalemma proton pump in the fusicoccin-dependent activation of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase) and of ethylene production in cultured Acer cells has been investigated. A good correspondence between the degree of acidification of the medium and the increase of ethylene production was observed for a wide range of stimulating fusicoccin concentrations. Vanadate and Erythrosin B, known as inhibitors of the plasmalemma proton pump, inhibited both the acidification of the medium and the stimulation of ethylene production; 0.6 M mannitol did not inhibit the «basal» production of ethylene either in hormone depleted cells or in cells re-supplied with growthpromoting 2,4-dichlorophenoxyacetic acid (2,4-D). On the contrary, mannitol practically suppressed the stimulation of ethylene production by fusicoccin but the acidification of the medium was only slightly reduced. Vanadate did not influence the cellular level of ACC. Mannitol almost doubled the level of ACC in the fusicoccin treated cells. The data are discussed in terms of regulation by the plasmalemma proton pump of an ACC oxidase presumably associated to the plasma membrane.

The effects of sugars on the development of hexose phosphorylating enzymes in the castor bean cotyledons

Abstract The rate of development of soluble glucokinase and fructokinase activities in cotyledons isolated from castor bean seeds germinated for 36–48 hr is markedly increased by the presence of glucose, fructose or sucrose in the incubation medium. A similar effect is found for galactose on galactokinase activity. A certain degree of specificity in the effects of the different sugars on the three enzymes is observed. The rates of development of other enzyme activities such as phosphoglucomutase, glucose-6-phosphate dehydrogenase, aldolase, and NADP-dependent isocitrate dehydrogenase are not affected by the sugars. It is suggested that the effects of the sugars on kinase activities are due to enzyme induction of the type described in bacteria and lower organisms.

Ferricyanide Induced Ethylene Production is a Plasma Membrane Proton Pump Dependent 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Oxidase Activation

Summary In sycamore cells ( Acer pseudoplatanus L.) the stimulation of ethylene formation dependent on the trans-plasma membrane (trans-PM) reduction of applied ferricyanide [K 3 Fe(CN) 6 ] was previously shown to be independent of applied 1-Aminocyclopropane-1-carboxylic acid (ACC) and was tentatively ascribed to an activation of ACC oxidase. In this paper we report that in ferricyanide stimulated cells: I) the cellular level of ACC does not change II) the activation of ACC oxidase is not prevented by 1 mM cycloheximide (CHE) and ill) extracts from control and treated cells possess the same amount of ACC oxidase activity. These data confirm the activation of ACC oxidase and indicate that it does not require de novo protein synthesis but is an increase of catalytic efficiency, which is lost in the cell extracts. Nutritional experiments with control and iron depleted cells indicate that it is not related to an increased availability of Fe ++ in the growth medium. Ethylene production correlates well with proton extrusion in the medium. Inhibitors of the plasmalemma proton pump used at concentrations only slightly inhibiting the rate of ferricyanide reduction strongly inhibited the development of ACC oxidase activation. Finally, activation of ACC oxidase was not induced in K + depleted cells but it was only delayed if K + was resupplied before or concomitantly to the ferricyanide reduction. The data are discussed in terms of control of ACC oxidase activity by the plasmalemma proton pump.

Eukaryotic N10-formyl-H4folate:Methionyl-tRNAf transformylase: Some properties of the Euglena gracilis enzyme

Abstract The N 10 - formyl-H 4 folate:methionyl-tRNA fMet transformylase was extracted from Euglena cells, and the level of enzyme activity found to be of 0.15 pmol/min per 106 cells for autotrophic and 0.016 pmol/min per 106 cells for streptomycin-bleached cells. The enzyme has been purified by DEAE-cellulose and CM-Sephadex chromatography about 1000-fold from autotrophic cells and about 650-fold from chloroplasts obtained by the non-aqueous technique. A methionyl-tRNA synthetase with apparently high specificity for the initiator tRNAMet has also been found which allowed the estimation of enzyme activity even with unfractionated tRNAs from different sources, since only formylatable species were present in methionylated form. As judged from the initial velocity the Euglena transformylase showed the same kinetic behaviour towards Met-tRNAfMet from E. gracilis, Escherichia coli and yeast. The chloroplast enzyme showed different properties from those of the bacterial enzyme. It appears to have a partial requirement for K+, a higher molecular weight, and a lower Mg2+ optimum. Moreover, the chloroplast transformylase showed sigmoidal rather than hyperbolic kinetics respect to Met-tRNAfMet ( n H = 1.81 ; K′ = 4 · 10 −14 ) as well as N 10 - formyl-H 4 folate ( n H = 2.46 ; K′ = 5.2 · 10 −16 ), thus indicating a strong positive cooperativity for both substrates.

Growth-dependent changes of folate metabolism and biosynthesis in cultured Daucus carota cells

Abstract Metabolism of folates has been studied in cultures of Daucus carota cells. With the addition to the culture medium of natural folates as folic acid, 5-HCO-H 4 PteGlu, 5-CH 3 -H 4 -PteGlu and pteroic acid the final yield was scarcely affected but folic acid stimulated the initial rate of growth. On the contrary, the growth rate was reduced by 5-HCO-H 4 -PteGlu. Growth was severely inhibited by folate analogues such aminopterin and methotrexate and by sulfanilamide, the inhibition of the latter being easily reversed by equimolar amount of folic acid. Microbiological assays of cell folates with Lactobacillus casei were performed at different stages of growth, covering the entire culture cycle. Most folates can be measured only after treatment of the extracts with conjugase (γ-glutamyl-hydrolase, an enzyme removing glutamate from the glutamate chain of folates) thus indicating an high ratio between poly- and mono-plus oligo-glutamate forms. Radiochromatographic analyses of folates obtained by cells cultured in [ 3 H]folic acid show the presence, besides of tri- and penta-glutamates, of forms with very long chains, of unidentified length. A strong shortening of the glutamate chain length was obtained with aminopterin and methotrexate. The ratio poly/mono, as well as the cell amount of folates, varies during the culture cycle. Rate of folate accumulation reaches its maximum before that of culture growth and sharply decreases before the slowing down of cell divisions, suggesting that depression or repression of folate biosynthetic pathways precedes the changes in the trend of cell replication.

Identification and cell level of folate derivatives from growing cultures of streptomycin-bleached Euglena gracilis

A general description of folate derivatives of growing cells of a streptomycin-bleached strain of Euglena gracilis Klebs strain Z Pringsheim has been obtained by combining data from differential microbiological assays on total or fractionated cell extracts with information from radiochromatographic analyses of folates from cells grown in [3H] folic acid or p-[14C]-aminobenzoic acid. Lactobacillus casei assays, after treatment of the Euglena extracts with purified γ-glutamyl-carboxypeptidase, gave a value of about 60 μg/g dry wt. of folic acid. The distribution of the various forms of folates was 1% as formyl-monoglutamate, 8.6% as formyl di- and triglutamate, 25.9% as formyl-polyglutamate, 1.5% as methyl-monoglutamate, 10.4% as methyl di- and triglutamate and 52.4% as methyl-polyglutamate. No folates with an oxidized pteridine ring were detected. The preferred polyglutamate form was the hexaglutamate. Cell content, ratio between formyl and methyl derivatives, and percentage distribution in the mono-, oligo- and polyglutamate forms are compared with those of other organisms or tissues.