Amino acid transport inRhodotorula glutinis

The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10⁻⁶ m l-lysine and 1500∶1 for 10⁻⁶ m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically. The influx is saturable with an apparentK _m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was 13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed a powerful “trans” inhibitory effect on the uptake of amino acids.     

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h^–1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.     

Effect of pH on inhibition of cyanide-resistant respiration by hydroxamic acids inRhodotorula glutinis

The extent of inhibition of cyanide-insensitive respiration inRhodotorula glutinis by salicylhydroxamic (SHAM) and bezhydroxamic acid depends on the pH of the cell suspension; maximum inhibition occurs at pH 4–6, at higher pH values the inhibitory capacity Tapidly drops. The acidity of the medium affects primarily the degree of dissociation of hydroxamic acids which, as weak acids, pass through the yeast cell membrane only as neutral molecules (Kotyk 1963; de la Peánaet al. 1982). In a medium more alkaline than the cell interior the fraction of the acids distributed into the cell decreases and a full inhibition requires an increased concentration of the acid in the medium. Cyanide-resistant respiration was also found inSaccharomyces cerevisiae; even here medium alkalinization eliminates the inhibitory effect of hydroxamic acids but, in contrast toR. glutinis, the cell membrane is permeable also to 2-iodobenzhydroxamie acid. Comparison of the effect of pH on the action of SHAM on cyanide-resistant respiration inR. glutinis, on the concentration of dissociated and undissociated form of SHAM in the intracelluler fluid and on its chelating ability refutes the notion that chelation of nonheme iron forms the basis of inhibiton of the alternative oxidase. The authors thank Dr. Karel Janáček, DrSc., for valuable comments concerning the evaluation of experimental results.     

tight coupling of monosaccharide transport and metabolism inRhodotorula gracilis

A variety of agents have been applied to separate the uphill, metabolically linked transport of monosaccharides from facilitated diffusion inRhodotorula gracilis. The substances used were iodoacetamide, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, sodium azide, sodium fluoride and heavy-metal cations, including Cd²⁺, Hg²⁺, Cu²⁺, UO²⁺ ₂, La³⁺ and Th⁴⁺. No agent examined led to such separation. There was always either a complete inhibition of both cell metabolism (or uncoupling of oxidative phosphorylation) and the transport mechanism or the transport of sugars uphill persisted (when the metabolism was only partly inhibited). It is concluded that the monosaccharide transporting system inRhodotorula gracilis is tightly coupled to cell metabolism and cannot operate without sufficient energy supply even in cases when no uphill movement is involved.     

Regulation of phosphate metabolism during intracellular lipid production inRhodotorula gracilis

Summary ³¹P NMR spectra of intact cells ofRhodotorula gracilis showed pH dependent inorganic phosphate (P_i) signals from two different compartments, namely, vacuole and cytoplasm. Clear distinction between growth phase and lipid accumulation phase was inferred by monitoring glycerol 3-phosphate and polyphosphate signals. The role of glycerol 3-phosphate in lipid production and the significance of polyphosphate accumulation during the same is discussed.     

Function of carbohydrate moiety in acid phosphatase of Rhodotorula glutinis

The properties of native and partially deglycosylated forms of acid phosphatase from Rhodotorula glutinis were compared. The removal of carbohydrate moiety resulted in higher thermostability and resistance to proteolysis whereas specific activity, pH optimum and Km value with p-nitrophenyl phosphate remained unchanged. The role of carbohydrate moiety in stabilization of the enzyme structure and protection against proteolysis is suggested.     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Some effects of nitrogen-starvation on nitrogen and carbohydrate metabolism inAnkistrodesmus braunii

Enzymic activities have been measured in cell-free extracts from nitrogen-starved cultures ofAnkistrodesmus braunii. During ten hours of nitrogenstarvation the activities of the enzymes nitrite reductase (E.C.1.6.6.4), glutamic dehydrogenase (E.C.1.4.1.4), glutamine synthetase (E.C.6.3.1.2) and urea amidolyase (E.C.3.5.1.5) were derepressed while the activities of the enzymes malate dehydrogenase (E.C.1.1.1.37) and hexokinase (E.C.2.7.1.1) remained more or less unchanged. In contrast, the photosynthetic capacity of the nitrogen-starved cultures declined rapidly and accompanying this decline were losses in the activities of ribulose diphosphate carboxylase (E.C.4.1.1.39) and triose phosphate-NADP-dehydrogenase (E.C.1.2.1.13).     

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.     

Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis

2-Deoxy-D-glucose transport by Rhodotorula glutinis is an active process. The intracellular concentration of free deoxyglucose after 15 min incubation of Rhodotorula cells with this sugar was 230 times the extracellular concentration. Although cell extracts at this time contained more 2-deoxy-D-glucose 6-phosphate than deoxyglucose, pulse-labelling experiments demonstrated that deoxyglucose is transported as the free sugar and subsequently phosphorylated. After transport, Rhodotorula cells metabolize deoxyglucose. The major metabolites during 30-90 min incubations were determined to be 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucitol, 2-deoxy-D-gluconate and 2,2'-dideoxy-alpha, alpha'-trehalose. Rhodotorula glutinis also degrades deoxyglucose to CO2. The concentrations of intermediates in this pathway were too low to detect and resolve in extracts of control cells. In 2,4-dinitrophenol-poisoned cells, however, it appears that deoxyglucose degradation is restricted largely to loss of C-1 as CO2 and it was possible to identify 1-deoxy-D-ribulose 5-phosphate as an intermediate presumably arising from metabolism of deoxyglucose by the oxidative portion of the hexose monophosphate pathway.