Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley (Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light.Pulses of [³⁵S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

Cytochemical localization of glycolate oxidase in microbodies of Klebsormidium

The filamentous green alga Klebsormidium flaccidum A.Br. was fixed with glutaraldehyde, incubated in a cytochemical medium designed to detect glycolate-oxidase activity, and prepared for electron microscopy. Heavy deposits of stain were observed in microbodies following incubation with either glycolate or L-lactate as substrate, but not after incubation with D-lactate or H₂O. When Chlamydomanas reinhardi Dangeared cells were treated in the same way, their microbodies did not appear stained. The results establish that in Klebsormidium glycolate-oxidase occurs in microbodies (peroxisomes), as it does in angiosperms; also, they emphasize the dichotomy between those green algae which contain glycolate-oxidase and those, such as Chlamydomonas, which possess the mitochondrial enzyme glycolate dehydrogenase.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Intracellular localization of certain photosynthetic enzymes in bundle sheath cells of plants possessing the C4 pathway of photosynthesis.

Bundle sheath cells were enzymatically isolated from representatives of three groups of C₄ plants: Zea mays (NADP malic enzyme type), Panicum miliaceum (NAD malic enzyme type), and Panicum maximum (phosphoenolpyruvate (PEP) carboxykinase type). Cellular organelles from bundle sheath homogenates were partially resolved by differential centrifugation and on isopycnic sucrose density gradients in order to study compartmentation of photosynthetic enzymes. A 48-h-dark pretreatment of the leaves allowed the isolation of relatively intact chloroplasts. Enzymes that decarboxylate C₄ acids and furnish CO₂ to the Calvin cycle are localized as follows: NADP malic enzyme, chloroplastic in Z. mays; NAD malic enzyme, mitochondrial in all three species; PEP carboxykinase, chloroplastic in P. maximum. The activity of NAD malic enzyme in the three species was in the order of P. miliaceum > P. maximum > Z. mays. There were high levels of aspartate and alanine aminotransferases in bundle sheath extracts of P. miliaceum and P. maximum and substantial activity in Z. mays. In all three species, aspartate aminotransferase was mitochondrial whereas alanine aminotransferase was cytoplasmic. Based on the activity and localization of certain enzymes, the concept for aspartate and malate as transport metabolites from mesophyll to bundle sheath cells in C₄ species of the three C₄ groups is discussed.     

Aryl hydrocarbon hydroxylase of rat brain mitochondria: Properties of,and effect of inhibitors and inducers on,enzyme activity

Aryl hydrocarbon hydroxylase (AHH), a typical example of mixed-function oxidase system, was studied in rat brain mitochondria. The enzyme was found to require oxygen and NADH for optimal expression of the activity. Coaddition of NADPH in the incubation system containing NADH resulted in an additive effect on the enzyme activity. NADH- and NADPH-dependent mitochondrial AHH activity was linear with respect to protein concentration and incubation time. The enzyme exhibited a sharp optima at pH 7.6. Specific activity of NADH-dependent mitochondrial AHH in rat brain was 3–4 and 8–11 times higher than that of NADPH-dependent mitochondrial and microsomal enzyme activity, respectively. Of the species investigated, NADH-dependent mitochondrial AHH followed the order: mice ? guinea pig > rat, while NADPH-supported mitochondrial AHH was in the order: rat > guinea pig ? mice. Specific activity of NADH-dependent mitochondrial AHH in various rat brain regions was similar with the exception of olfactory lobes which exhibited 60% higher activity than other region. When total region activities were added approximately whole brain activity was recovered. The apparent K_m value of NADH-dependent mitochondrial AHH was 1.18 μm with benzo(a)pyrene as a substrate. This K_m value was five to six times lower than that of NADPH-dependent microsomal AHH in rat brain (6.66 μm). NADH-dependent mitochondrial AHH was inhibited by KCN in a concentration-dependent manner while NADPH-supported mitochondrial AHH did not reveal any sensitivity to cyanide. Brain microsomal NADH as well as NADPH-supported AHH was also inhibited by KCN in a concentration-dependent manner. Carbon monoxide inhibited NADH-dependent mitochondrial AHH activity (48%) and had no effect on NADPH-dependent mitochondrial enzyme. Mitochondrial NADH and NADPH-dependent AHH activities were induced by 3-methylcholanthrene (64–73%) and benzo(a)pyrene (91–92%) pretreatments while no induction occurred with phenobarbital administration. 1-Benzylimidazole, SKF 525 A, metyrapone, and α-naphthoflavone inhibited both basal and 3-methylcholanthreneinduced NADH-dependent mitochondrial AHH activity. α-Naphthoflavone was more effective in inhibiting 3-methylcholanthrene-stimulated rat brain NADH-dependent mitochondrial AHH. Mitochondrial NADH-dependent AHH activity increased gradually with the onset of development and attained a steady state after 49–56 days of age. An increase of eight- to ninefold in the specific enzyme activity was observed between 7- and 56-day-old rats. No significant increase in brain mitochondrial AHH activity was observed between 56- and 91-day-old rats.     

Identification and Characterization of MAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

Stimulation of delta-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA

Formation of the chlorophyll and heme precursor δ-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond formation. Our results suggest that the RNA that is required for ALA formation may be functionally distinct from the glutamyl-tRNA species involved in protein synthesis.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation

The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark-grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis.     

Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

Microbody enzymes and carboxylases in sequential extracts from c(4) and c(3) leaves

A seven-step sequential grinding procedure was applied to leaves of Atriplex rosea, Sorghum sudanense, and Spinacia oleracea to study the distribution of carboxylases and microbody enzymes. In the extracts from C₄ species there were 7- to 10-fold reciprocal changes in specific activities of ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase. No such changes occurred in sequential extracts from spinach. No inhibitors of ribulose-1, 5-diphosphate carboxylase were detected when the mesophyll extracts of Sorghum were assayed together with spinach extracts. These results reaffirm the conclusion of others that phosphoenolpyruvate carboxylase is largely confined to the mesophyll in these species and ribulose-1, 5-diphosphate carboxylase to the bundle sheath. The specific activities of glycolate oxidase and hydroxypyruvate reductase in bundle sheath extracts were two to three times those in mesophyll fractions. Catalase behaved similarly in Atriplex rosea but in Sorghum the specific activity was virtually the same in all fractions. From the relative amounts of these enzymes present, and comparison with the data obtained from spinach, it is concluded that typical leaf peroxisomes are present in the bundle sheaths of both C₄ species and in the mesophyll of Atriplex rosea. The relative enzyme activities in the mesophyll of Sorghum suggest that the microbodies there are of the non-specialized type found in many nongreen tissues. The activities of the microbody enzymes in the bundle sheath of Sorghum seem quite inadequate to support photorespiration.     

Induction of Catalase Activity in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

The methanol-grown cells of Kloeckera sp. No. 2201 exhibit a markedly high catalase activity as compared with the glucose-grown and ethanol-grown cells. In this connection, specific organelles (“microbodies”) appear only in the methanol-grown cells. When the yeast cells harvested from a methanol medium (cells whose catalase activity had been enhanced to an appreciable extent: “partially induced cells”) were transferred into media containing glucose, ethanol or methanol as the sole carbon and energy source, further increase of catalase activity was mediated only by methanol. This induction of catalase activity was partially inhibited by cycloheximide at its high concentration, but chloramphenicol did not show any effect. Glucose inhibited strongly the induction by methanol, while galactose gave no effect. Electron microscopical observation revealed that the development of microbodies in the cells growing on methanol was hardly affected by cycloheximide. Disappearance of microbodies was observed electron microscopically after the methanol-grown cells (partially induced cells) were transferred to a methanol-glucose medium and cultivated for 8 hr. 3′,5′-Cyclic AMP or dibutyryl-3′,5′-cyclic AMP could not eliminate the inhibitory effect of glucose on the catalase induction. Addition of caffeine or theophylline did not promote the action of the cyclic nucleotides. 3-Amino-1,2,4-triazole inhibited only 40% of the hydrogen peroxide-decomposing activity in the cell homogenate of methanol-grown cells even at its concentration of as high as 10 mm, while sodium azide inhibited the enzyme activity completely at the concentration of 1 mm.     

β-oxidation enzymes and the carnitine-dependent oxidation of palmitate and palmitoyl CoA in mitochondria from avocado

Two sites for the β-oxidation of fatty acids in avocado (Persea americana L.) mesocarp exist. One site is the microbody, the other the mitochondrion. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose density gradient centrifugation, prevents rapid access of acyl CoA substrates to matrix β-oxidation sites. Thus, intact mitochondria showed little β-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of the acyl CoA substrates to matrix sites. Consequently, in ruptured mitochondria, high O₂-oxidation enzyme activities were measured. O₂ uptake studies further distinguished the two organellar sites of β-oxidation. During palmitoyl CoA oxidation, O₂ uptake was reduced by catalase and increased by KCN in the microbodies, whilst mitochondrial O₂ uptake was unaffected by catalase and reduced by KCN. This reflected the differing fates of FADH₂, produced during the first β-oxidation step, in the two organelles. In addition, only the mitochondrial β-oxidation of fatty acids was carnitine-dependent.