Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.

Glucose-initiated germination of mucor racemosus sporangiospores.

Treatments leading to the initiation of germination of Mucor racemosus sporangiospores were examined. The results support the hypothesis that glucose is a specific trigger molecule for the initiation of Mucor racemosus sporangiospores. Glucose and some of the glucose analogues tested could initiate germination, mannose, 3-O-methyl-D-glucose, 5-thio-D-glucose and 6-deoxy-D-glucose being the most effective. The initiation event appeared to depend on the concentration of the initiator, with glucose and 3-O-methyl-D-glucose exhibiting nearly identical kinetic constants. Spores accumulated not only glucose and 3-O-methyl-D-glucose, but also the 1-O-methyl-D-glucose analogue, which did not initiate germination. The accumulated 3-O-methyl-D-glucose was not metabolized. The initiation sequence appeared to require the continued presence of the initiator as well as protein synthesis.

Role of amino acids and endogenous protein in the germination of Mucor racemosus sporangiospores.

The role of amino acids as triggers of Mucor racemosus sporangiospore germination was investigated. No single amino acid was effective as glucose or peptone at triggering germination. Germination induced by glucose or peptone was pH-independent, whereas germination induced by glutamate was pH-dependent. The composition of the free amino acid pools of M-spores (those unable to germinate on glutamate) was qualitatively and quantitatively similar to that of C-spores (those capable of germinating on glutamate) with the exceptions of hydroxyproline and methionine and methionine whose concentrations were several-fold higher in C-spores. Glutamate and leucine were taken up by germinating and nongerminating spores; however, significant protein synthesis occurred only in germinating spores. Spores not triggered by 3-O-methyl-D-glucose (M-spores) contained about one-half the protein of those triggered by 3-O-methyl-D-glucose (C-spores). C-spores initiated to germinate by 3-O-methyl-D-glucose decreased in total organic carbon and protein over a 6 h period; removal of the 3-O-methyl-D-glucose resulted in an immediate halt of protein degradation and spore swelling. These results suggest that protein is a major endogenous reserve in M. racemosus sporangiospores and that its turnover is a necessary event for glucose-triggered germination.

Relationship between sporulation medium and germination ability of Mucor racemosus sporangiospores.

Asexual sporangiospores of Mucor racemosus produced on a minimal sporulation medium (M spores) germinated only if glucose, mannose or a complex substrate such as peptone, yeast extract or Casamino acids was present. Once germinated, growth was supported by a wide range of substrates including amino acids, carbohydrates or organic acids. Sporangiospores produced on a nutritionally complex sporulation medium (C-spores) germinated on a wide range of carbon sources. C-spore phenotype was pleiotropic in that sporangiospores capable of germinating on cellobiose could always germinate on glutamate or xylose; but C-spores capable of germinating on xylose or glutamate did not always germinate on cellobiose. There was a hierarchy of substrates capable of initiating germination with glucose = mannose greater than xylose greater than glutamate greater than cellobiose. C-spores also differed from M-spores by initiating germination in the presence of the non-metabolizable glucose analogue 3-O-methylglucose. These results suggest that at least two sporangiospore phenotypes are produced depending upon the concentration and type of ingredients present in the sporulation medium.

Glyoxylate cycle in Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was grown in media containing acetate, glutamate, and peptone as carbon sources. The component enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were present under these conditions throughout the growth cycles. Highest specific activities for each enzyme were found in media with acetate as the carbon source. In an enriched peptone medium containing glucose, neither activity was detected until glucose was exhausted from the medium. Treatment of acetate-grown cells with glucose resulted in a rapid decline in the specific activities of both enzymes. The importance of this cycle in acetate-grown cells was indicated by the ability of itaconic acid (100 mM) to inhibit the growth of M. racemosus in acetate but not glutamate media. Itaconate was also shown to be a potent inhibitor of isocitrate lyase activity in vitro.

Purification and properties of a triacylglycerol lipase from Mycobacterium phlei.

In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified 800-fold from stationary phase cells of Mycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, pI. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. ith phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent Km of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested (C2-C18), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p=nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.

Pyruvate kinase isozymes of Mucor racemosus: control of synthesis by glucose.

A variety of cultural conditions were examined to determine the relationship between pyruvate kinase isozyme patterns and morphology in Mucor racemosus. The results indicate that M. racemosus has two isozymes of pyruvate kinase, form A and form B, which are clearly separable on ion-exchange columns (diethylaminoethyl-cellulose). Addition of glucose to cultures growing on amino acids in air resulted in the induction of form A and the termination of form B synthesis. Cycloheximide added at the same time as glucose blocked the formation of form A but did not interfere with the termination of form B synthesis. Removal of glucose resulted in termination of form A synthesis and the induction of form B. Cycloheximide blocked the induction of form B and did not interfere with the termination of form A synthesis. The data show that the isozyme type is not directly related to morphology, but depends only on the presence or absence of glucose.

Respiratory capacity, cyclic adenosine 3',5'-monophosphate, and morphogenesis of Mucor racemosus.

A variety of cultural conditions were examined to determine the relationship between respiratory capacity and the growth of Mucor racemosus in the yeast and mycelial form. The results show that both yeasts and hyphae can develop when the respiratory capacity is low (e.g., in N2). In addition, the yeast form of the fungus could be grown in air in the presence of cyclic adenosine 5'-monophosphate with high respiratory rates characteristic of air-grown mycelia. These results indicate that their is not an obligatory relationship between respiratory capacity and morphogenesis in M. racemosus. Low intracellular levels of cyclic adenosine 5'-monophosphate, however, were correlated with aerobic mycelial development, whereas yeast development under CO2 was characterized by higher cyclic adenosine 5'-monophosphate levels.