Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth⁺) in the absence of catalase activity. These Mth⁺ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H₂O₂ does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H₂O₂-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized. Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997     

Production of Catalases by Aspergillus niger Isolates as a Response to Pollutant Stress by Heavy Metals

Isolates of Aspergillus niger, selected from the coal dust of a mine containing arsenic (As; 400 mg/kg) and from the river sediment of mine surroundings (As, 1651 mg/kg, Sb, 362 mg/kg), growing in minimal nitrate medium in the phase of hyphal development and spore formation, exhibited much higher levels of total catalase activity than the same species from the culture collection or a culture adapted to soil contaminated with As (5 mg/L). Electrophoretic resolution of catalases in cell-free extracts revealed three isozymes of catalases and production of individual isozymes was not significantly affected by stress environments. Exogenously added stressors (As⁵⁺, Cd²⁺, Cu²⁺) at final concentrations of 25 and 50 mg/L and H₂O₂ (20 or 40 mM) mostly stimulated production of catalases only in isolates from mines surroundings, and H₂O₂ and Hg²⁺ caused the disappearance of the smallest catalase I. Isolates exhibited a higher tolerance of the toxic effects of heavy metals and H₂O₂, as monitored by growth, than did the strain from the culture collection.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Glutamine synthetase isoenzymes, oligomers and subunits from hairy roots of Beta vulgaris L. var. lutea

A cytosolic and a plastidic isoenzyme of glutamine synthetase (GS; EC 6.3.1.2) were separated from hairy roots of Beta vulgaris L. var. lutea. The predominant activity was that of cytosolic GS 1; the relative proportion of plastidic GS 2 activity changed, however, depending on the growth conditions. Maximum activity of both isoenzymes was measured after growth with NO⁻ ₃ as the major N-source. Growth with NH⁺ ₄ as the sole N-source or growth in constant darkness resulted in a significant decrease in GS 1 activity, whereas GS 2 activity was much less effected and thus contributed as much as 25% of total root GS activity. The isoenzymes GS 1 and GS 2 were active both in the octameric and tetrameric states. Both oligomers of GS 2 and octameric GS 1 were active under all growth conditions applied whereas tetrameric GS 1 was not active when the roots were grown under light-dark changes with NO⁻ ₃ as the major N-source. The molecular masses of the subunits were identical for both isoenzymes. Glutamine synthetase 1 was composed of up␣to four different 38-kDa subunits and two different 41-kDa subunits; GS 2 was assembled from one type of 38-kDa subunit and one type of 41-kDa subunit. The GS␣2 subunits were most probably identical to two of the GS␣1 subunits. The subunit composition of GS 1, but not of GS 2, changed depending on the growth conditions of the roots. Changes in GS 1 subunit composition were correlated with changes in GS 1 activity. The different growth conditions induced the specific assembly of different GS 1 isoenzymes which could, however, not be separated by anion-exchange chromatography but became evident only after two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Received: 30 May 1997 / Accepted: 28 August 1997     

A Proposal for the Expression of the Foamability of Protein Solutions

Mutants exhibiting high catalase activity were derived from Candida boidinii S2 strain AOU-1, from among mutants resistant to H₂O₂, NaN₃ or 3-amino-1,2,4-triazole (ATA). The catalase activity of an ATA-resistant strain was improved by means of a methanol-limited chemostat culture with H₂O₂ supplementation. The catalase activity increased with increasing H₂O₂ concentration in the feed medium in the range where methanol did not remain. Alcohol oxidase activity increased after adaptation of the cells to H₂O₂. Cells of mutant strain SA051 grown under the optimal culture conditions produced 1200 mm formaldehyde in the reaction mixture.     

Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response

When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H₂O₂, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K _m of 40 mM and a V _max of 285,000 U (mg protein)^–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases. Received: 15 October 1997 / Accepted: 2 February 1998     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Regulation of catalase enzyme activity by cell signaling molecules

Mitogenic cell proliferation requires a rapid and transient H₂O₂ generation, which is blocked by catalase or PKA activators. Previously, we observed that anemic HIV(+) individuals expressed acidic pIs of catalase in RBC with significantly high activities [Mol Cell Biochem 165: 77–81, 1996]. These findings led us to hypothesize that cell signaling molecules regulate catalase to control cell mitogenesis. To test the hypothesis, we determined (i) whether RBC counts correlate with their catalase activities, (ii) whether protein kinases and phosphatases alter catalase activity in vitro, and (iii) whether protein kinase activators increase catalase activity to suppress proliferation of cultured cells. The results indicated that RBC counts inversely correlated with RBC catalase activities in both HIV(+) (r: –0.6769, r²: 0.4582, n: 69 male, p < 0.0001) and HIV(–) (r: –0.3827, r²: 0.1464, n: 177 male, p < 0.0001) populations. Catalytic PKA, PKC and Casein Kinase II, but none of PKG, Ca²⁺/calmodulin kinase II and p34cdc/cyclinB, rapidly elevated catalase activity in vitro by up to 2-fold. Whereas a major CAT subunit (60 kDa) showed immunoreactive phosphoserine and phosphothreonine, the kinases- and -³²P-ATP-dependent phosphorylation occurred with a minor component (110 kDa). Among PKC isozymes examined, PKCz was the most effective modulator followed by PKC, and protein phosphatase 1 and 2A decreased the catalase activity. PKA and PKCz activators of forskolin and okadaic acid increased catalase activity and 110 kDa expression in NIH3T3 cells up to 2.4-fold and suppressed the cell growth, showing an inverse correlation of the indices (r: –0.9286, r²: 0.8622, n: 18, p < 0.0001). Taken together, these results suggest for the first time that catalase is under the regulation of cell signaling molecules and capable of modulating mitogenic cell proliferation.     

Two δ1-pyrroline-5-carboxylate dehydrogenase isoforms are expressed in cultured Nicotiana plumbaginifolia cells and are differentially modulated during the culture growth cycle

δ¹-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl⁻ at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K _m values for the two substrates, P5C and NAD⁺, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed. Received: 23 May 1996 / Accepted: 18 December 1996     

Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae

The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition. This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium. S. cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids. Inhibition of cell division in the presence of Cd(NO₃)₂ was accentuated by growth in the presence of a polyunsaturated fatty acid. Furthermore, susceptibility to Cd²⁺-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation. Thus, during exposure to Cd²⁺, K⁺ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells. In addition, reductions in cell viability during exposure to Cd²⁺ were most marked in polyunsaturated-fatty-acid-supplemented cells. At certain times, unsupplemented Cd²⁺-exposed cells displayed up to 7-fold greater viability than supplemented Cd²⁺-exposed cells. The study demonstrates that the toxicity of the redox-inactive metal Cd²⁺ towards S. cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation. Received: 14 March 1997 / Received revision: 4 June 1997 / Accepted: 7 June 1997     

Isolation and preliminary characterization of mutants of the cyanobacterium Nostoc muscorum resistant to growth inhibition by methylamine

Summary The wild-type heterocystous and nitrogen-fixing (Het ⁺ Nif ⁺) N. muscorum and its non-heterocystous non-nitrogen-fixing (Het ^- Nif ^-) mutant strain both fail to grow in different inorganic nitrogen media containing 1 mM methylamine hydrochloride (MA). Mutants of the Het ⁺ Nif ⁺ and Het ^- Nif ^- parents resistant to growth inhibition by 5 mM MA and thus designated as MA ^R strains were isolated with a frequency of 2.5(±2.4)×10⁶. A MA ^R strains of the Het ⁺ Nif ⁺ and a MA ^R strain of the Het ^- Nif ^- parent were characterized for growth, heterocyst formation and acetylene reducing activity in the presence and absence of methylamine in N₂ medium. The Het ⁺ Nif ⁺ MA ^R strain grows better in MA containing than in MA-free N₂ medium, and all cultures grown with MA are found to lack both acetylene reducing activity and heterocyst. The Het ^- Nif ^- MA ^R strain shows good growth in MA-containing N₂ medium but no growth in MA-free N₂ medium. Furthermore, both the Het ⁺ Nif ⁺ MA ^R and Het ^- Nif ^- MA ^R strains show better growth in the presence than in the absence of MA in NO ₃ ^- and NH ₄ ⁺ media. These results appear to suggest that the MA ^R phenotype in N. muscorum is due to the metabolic utilization of the ammonium analog as a nitrogen source.     

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Increase of Copper Toxicity to Growth of Chlorella vulgaris with Increase of Light Intensity

Abstract In the absence of inhibitory concentrations of copper, the photoautotrophic growth of Chlorella vulgaris INETI58C at 27°C exhibited a higher specific growth rate and reached a higher maximal concentration of biomass, under irradiance of 150 W m⁻², compared with 100 W m⁻². However, when the mineral growth medium was supplemented with CuSO₄ (range 40–80 μM), algal growth was significantly affected at the higher light intensity. In the presence of Cu²⁺, the increase in dry biomass was uncoupled from the increase in cell concentration since more than 16 autospores gathered together, inside the enlarged mother cell, suggesting that copper arrested the normal bursting of the mother cell wall. At the higher irradiance, growth medium supplementation with 80 μM of CuSO₄ led to bleaching of photosynthetic pigments. No growth was observed, while, under the lower irradiance, growth was only slightly inhibited. Results clearly showed that copper toxicity to growth of C. vulgaris was strongly influenced by light intensity. Higher light intensity elicits lethal or sublethal Cu²⁺ damage at concentrations lower than the threshold level for damage at lower light intensities. Cu²⁺ may elicit lethal or sublethal light damage at irradiances lower than the threshold level for unpolluted aquatic systems. Received: 17 January 1997; Accepted 15 April 1997     

Effect of NaCl and CaCl<Subscript>2</Subscript> on the antioxidant mechanism of leaves and stems of the rootstock CAB-6P (<Emphasis Type="Italic">Prunus cerasus</Emphasis> L.) under in vitro conditions

The effect of salinity on the non-enzymic and enzymic antioxidant activity, shoot proliferation and nutrient accumulation was studied in in vitro cultures of the rootstock CAB-6P (Prunus cerasus L.). Three concentrations (0, 30 and 60 mM) of NaCl or CaCl₂ were added to a modified MS medium. Between the two salt treatments used, only the explants treated with CaCl₂ presented significant decrease in growth parameters. The concentrations of Na⁺ and Cl⁻ in the explants treated with NaCl were increased, as NaCl in the culture medium increased. Furthermore, in the explants treated with CaCl₂ the concentrations of Ca²⁺ and Cl⁻ were increased while that of K⁺ decreased, as CaCl₂ concentration increased. The activity of peroxidase in leaves as well as the number of its anionic isoforms was increased under 30 mM CaCl₂ as well as 60 mM NaCl or CaCl₂. On the contrary, increasing salinity, from 0 to 60 mM CaCl₂, resulted in a reduction of the catalase activity in leaves followed by disappearance of the only one catalase isoform that was detected in leaves (60 mM CaCl₂). In the stems of the explants treated with NaCl the peroxidase activity was reduced. In the stems and leaves of the explants grown in saline substrate the non-enzymic antioxidant activity was significantly increased. The results suggest that the stems and leaves of CAB-6P explants presented variable antioxidant responses that were depended on the salt form used. The contribution of enzymic and non-enzymic protection mechanisms to the adaptation of CAB-6P explants under salinity stress is discussed.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

c-myc-dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors

Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H₂O₂). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H₂O₂-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion. J. Cell. Physiol. 170:192–199, 1997. © 1997 Wiley-Liss, Inc.     

Catalase Activities of Phanerochaete chrysosporium Are Not Coordinately Produced with Ligninolytic Metabolism: Catalases from a White-Rot Fungus

Phanerochaete chrysosporium uses hydrogen peroxide as a substrate in its ligninolytic phase. To determine how the fungus protects itself against detrimental effects of reactive oxygen species, catalase activity was examined. The fungus produced up to four different catalase isozymes, Cat A to D. Isozyme CatC was the dominant activity in all growth conditions. A minor catalase, CatD, was apparent in low-carbon culture and upon exposure of mycelium grown on high-nitrogen medium with 5–50 mm of hydrogen peroxide. In low-nitrogen culture, an increase in catalase-specific activity preceded the onset of ligninolysis by 3 days. In low-carbon culture, catalase was produced at an even higher level without development of ligninolysis. Thus, catalase activity was not coordinately produced with ligninolytic metabolism. Received: 23 May 2000 / Accepted: 21 July 2000     

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Summary The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm³ and turned out to be significant (t=7.8>4.04=t _5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D₂) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D₂O during the transition period and the density of enzyme from cotyledons grown on H₂O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D₂O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.     

Purine nucleoside phosphorylase polymorphism in sheep erythrocytes

A polymorphism of purine nucleoside phosphorylase is described in sheep erythrocytes. Two isozymes were distinguished electrophoretically, one with high activity (NP-1) and one with low activity (NP-2). Breeding data suggest that the two isozymes are the product of two codominant alleles, NP ¹and NP ². The K _m 's for inosine did not differ between NP-1 and NP-2; however, NP-2 had a lower pH optimum and was relatively unstable when incubated at 48 C.Contribution No. 421-J, from the Department of Pathology, Kansas Agricultural Experiment Station, Manhattan, Kansas. Supported in part by USPHS Grants HL-70119 and HL 12072.