Catalase formation in Rhodotorula mucilaginosa in relation to the effect of glucose and antibiotics

The developmental pattern of the catalase activity in Rhodotorulamucilaginosa, an obligate aerobe, was investigated in relationto its growth. The pattern of catalase activity does not runin a manner comparable with that of a respiratory capacity,because catalase activity takes a continual rise after the middleof the logarithmic growth, while a respiratory pattern runsa constant level during the corresponding growth phase. Additionof antimycin A to cells with a minimum catalase activity doesnot block the increase in the catalase activity. Chloramphenicoldoes not exert any recognizable effect on the catalase formationwhereas cycloheximide does create an intense inhibitory effect,regardless of addition times on the course of growth. Theseresults show that the synthesizing sites of yeast catalase aredifferent from mitochondria. ¹Present address: Department of Biology, Japan Women's University,Tokyo, Japan (Received February 18, 1970; )     

Some properties of NAD-independent {alpha}-glycerophosphate dehydrogenase of yeast

NAD-independent, mitochondrial -glycerophosphate dehydrogenaseof baker's yeast, Saccharomyces cerevisiae, was liberated fromcells and its nature was examined. Hydrogen acceptors, pH optimaand reaction rates with substrate and hydrogen acceptor of theenzyme were determined. A naturally occurring phenolic pigmentextracted from yeast cells was also found to function as aneffective hydrogen acceptor for the enzyme. Addition of FMNor FAD to the -glycerophosphate oxidation system largely acceleratedenzymatic activity, whereas the enzyme system was strongly blockedby SH-reagents. This suggests that the SH-group functions atan essential site. Clear-cut inhibition by antimycin A of electrontransfer to cytochrome c suggests the intermediation of cytochromeb. (Received December 13, 1968; )     

A PHENOLIC PIGMENT REACTIVE TO L-LACTATE CYTOCHROME C REDUCTASE OF YEAST

1. A phenolic pigment was extracted from baker's yeast. The pigmentis slowly autooxidizable, and rapidly oxidized with Rhus-laccaseor polyphenol oxidase and reduced by dithionite.
2. The pigmentdissolved in ethylether had an absorption peak at258 mµ,shoulders at 289 and 382 mµ and a plateauat 450–500mµ. The difference spectrum between oxidizedand reducedforms of the pigment showed a wide plateau around500 mµ.
3. The pigment supported the oxygen uptake by reconstructed enzymesystem: L-lactate, L-lactate cytochhrome c reductase and Rhuslaccaseor polyphenol oxidase. In its absence, no oxygen uptake tookplace. The pigment was replaced successfully with p-quinone,catechol and menadione, but not with ubiquinone. The sequenceof hydrogen transport can be represented: L-lactate L-lactatecytochrome c reductase "phenolic pigment" oxidase oxygen.

(Received August 11, 1967; )     

Respiratory aspects of Rhodotorula mucilaginosa

The absorption spectrum of intact cels of Rhodotorula mucilaginosaexhibits three absorption peaks for carotenoids and a Soretband for cytochrome(s). The difference spectrum between thereduced and oxidized states indicates there are components ofcytochrome b, c(c₁) and a+a₃. However, the respiratory aspectof this strain differs significantly from that of baker's yeast,Saccharomyces cerevisiae. The respiratory capacity is strikinglyenhanced in the transition from the lag to the logarithmic phaseof growth, and is accompanied with a special increase in cytochromeoxidase. Addition of TMPD (plus ascorbate) in the presence ofglucose barely affects oxygen consumption of the cells duringthis period, whereas it markedly affects consumption in oldercells. Sodium azide slightly inhibits respiration in the courseof growth, except during this limited transitional period. Azide-insensitiverespiration is also seen in the mitochondrial fraction preparedfrom cells grown to the stationary state. A similar situationis found with antimycin A. Morphological investigations were carried out electron-microscopicallyon the cells of different growth stages. (Received September 16, 1969; )     

Development of catalase activity in yeast in relation to growth and respiration

1. 1) When yeast cells grown anaerobically were adapted to aerobicculture in a normal medium, catalase formation was markedlyenhanced after the earlier stage of exponential growth of thecells. The same thing occurred with cells transferred from ananaerobic culture into a nitrogen deficient medium.
2. 2) Thecatalase activity of aerobically grown cells declinedprogressivelyuntil glucose, which had been added to the mediumwas profoundlyexhausted. This decline was followed by a progressiverecoveryof activity to a normal level with the growth of thecells.Similar behavior of catalase was also seen at low concentrationsof glucose, except that an abrupt rise in activity was observedat the beginning of incubation. Even when cells which had declinedto a minimum of catalase activity were aerated in phosphatebuffer, they continued to synthesize catalase.
3. 3) The patternof alteration of catalase activity during cellgrowth was accompaniedby a comparable pattern of alterationin respiratory capacity.On the basis of this finding, togetherwith the fact that antimycinA causes intensive depression incatalase formation, it maybe inferred that the formation ofthe respiratory chain conductsthe formation of catalase.
4. 4) In the presence of ethyl alcoholas the carbon source inplace of glucose, a rise in both catalaseactivity and respiratorycapacity occurred from initiation ofincubation. This fact canbe interpreted to mean that the repressiveeffect of glucoseon catalase formation depends on the aerobiccharacter of thecells.

(Received February 26, 1968; )     

STUDIES ON LACTATE CYTOCHROME C REDUCTASES OF YEAST WITH SPECIAL REFERENCE TO ELECTROPHORESIS ON POLYACRYLAMIDE GEL

1. Two lactate dehydrogenases, L(+)- and D(–)- lactate cytochromec reductase, were extracted from the baker's yeast after disintegrationof the cells by a FRENCH press. They are separated by electrophoresison polyacrylamide gel and their activities were compared bycolor density of formazan, the reduction product of nitrobluetetrazolium.
2. The ratio of L-lactate cytochrome c reductaseactivity to D-lactatecytochrome c reductase activity variedto a great extent, dependingon culture conditions. L-Lactatecytochrome c reductase waspredominant in resting cells; thereverse was the case withcells in early exponential stage ofthe growth.
3. When the cells in exponential stage of growthwere aerated withoutnitrogen source, there occurred an intensiveincrease of L-lactatecytochrome c reductase, accompanied bythe decrease of D-lactatecytochrome c reductase.
4. Effectsof inhibitors on the activity ratio of these two enzymeswereinvestigated. o-Phenanthroline, dinitrophenol, sodium azide,chloramphenicol, British antilewisite and antimycin A favored,in this order, the formation of L-lactate cytochrome c reductase.

(Received August 18, 1966; )     

STUDIES ON OXALIC ACID OXIDASE IN GREEN LEAVES

1. The leaves of Chenopodium ambrosioides L. were found to havean intense activity of oxalic acid oxidase. The enzyme was locatedin the chloroplast, being firmly hound to its structure. 2. Properties of this chloroplast oxalic acid oxidase were described.The strict aerobic nature and the stoichiometry of the reactionwere confirmed. 3. Isolation of the enzyme from chloroplasts was performed,rupturing the chloroplasts with a French pressure cell or usingpyridine-water (1:1) as an extracting medium. 4. The enzyme was found to contain flavine and its activitywas enhanced in the presence of flavine added. Accordingly,the enzyme was inferred to be a flavine enzyme. (Received December 23, 1963; )