John E. Gander

On the enzymic synthesis of lactose-1-PO4.

Abstract Evidence is presented that enzyme fractions obtained from bovine mammary tissue will catalyze the synthesis of lactose 1-phosphate from UDPG and glucose 1-phosphate according to the reactions: UDPG UDPGal UDPGal + G-1-P 1-phosphate + UDP Using radioactive glucose, glucose 6-phosphate, and doubly labeled glucose 1-phosphate in the presence of UDPG and the proper fortifiers, it was shown that neither glucose nor glucose 6-phosphate would act as the galactosyl acceptor while glucose 1-phosphate would function as the acceptor. The phosphorylated sugar formed yielded equal amounts of lactose and inorganic phosphate upon mild acid hydrolysis and gave a negative reducing test in alkaline solution. It migrated more slowly than glucose 1-phosphate in paper electrophoresis and could be separated from glucose 1-phosphate by means of ion-exchange chromatography if the reaction medium was first treated with phosphoglucomutase to convert the glucose 1-phosphate to glucose 6-phosphate. When the lactose phosphate was enzymically synthesized from glucose 1-phosphate doubly labeled with C 14 and P 32 and UDPG, no radioactivity could be found in the galactose moiety. The glucose moiety of the lactose contained approximately the same specific activity as the substrate glucose 1-phosphate, and the ratio of the specific activities of C 14 to P 32 was the same in the glucose and phosphate of the lactose phosphate as in the starting glucose 1-phosphate. The primary nucleotide formed was identified as UDP by paper chromatography.

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides.

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5-diphosphate (5ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5 ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.

Isolation and partial characterization of the exocellular polysaccharides of Penicillium charlesii: III. Heterogeneity in size and composition of high molecular weight exocellular polysaccharides☆

Abstract Extracellular polysaccharides of the fungus Penicillium charlesii G. Smith, fractionated on DEAE-Sephadex and Sephadex G-50, were further resolved on Sephadex G-100. Specific enzyme assays for glucose, galactose, and mannose and analysis using gas-liquid chromatography revealed that the most highly resolved preparations contained only these three hexoses in significant amounts and were continuously heterogeneous with respect to size and composition. The highest molecular weight portion was shown to be a phosphopolysaccharide. The ratio of anhydrohexose:P decreased as the particle size decreased. Sedimentation-rate studies supported gel-filtration experiments in demonstrating gross differences in size within a single fraction isolated from DEAE-Sephadex.

The appearance of exo-?-mannosidase in cultures of Penicillium charlessi

SummaryGrowth of the fungus Penicillium charlesii G. Smith on glucose, minimal salts medium results in the appearance of α-d-mannopyranosidase activity capable of hydrolyzing p-nitrophenyl-α-d-mannopyranoside. No activity is found until depletion of the carbon source, after which the enzyme activity rapidly increases in the mycelium. Prolonged incubation of the culture results in the appearance of small amounts of α-mannosidase activity in the growth medium. The initial release of a mannose-containing polysaccharide into the medium precedes the appearance of α-mannosidase by several days.

Glutathione synthetase of bovine lens: Anomalies of the enzyme-catalyzed formation of ophthalmic acid

The activity of glutathione synthetase from bovine lens was examined as a functions of the concentration of L-gamma-glutamyl-L-alpha-aminobutyrate, the dipeptide substrate required in the formation of ophthalmic acid. Several significant anomalies of the glutathione synthetase-catalyzed formation of ophthalmic acid were found. Curvilinearity of double reciprocal plots occurred with this substrate; this curvilinearity shows substrate activation of the reaction which is likely a result of negative cooperativity. Both ATP4- and, to a lesser extent Mg2+ inhibited the reaction, whereas MgATP2- is the substrate; maximum activity occurred with 2 mM Mg2+ in excess of the concentration of added ATP. This investigation shows that it is necessary to establish a defined set of conditions for reporting enzyme activity and that the usual practice of using very large concentrations of Mg2+ relative to ATP, and 5- to 20-fold excess of the dipeptide will give less than optimum activity. The unit of enzyme activity is suggested to be that activity in ml using 2 mM ATP, 4 mM Mg2+, 30 mM glycine and 15 mM L-gamma-glutamyl-alpha-aminobutyrate, which results in the formation of 1 nmole/minute of ADP or P(i). In this study, 5-AMP was for the first time, shown to be an inhibitor of the reaction with a K(i) of 0.9 mM.

In vivo biosynthesis of peptidophosphogalactomannan in Penicillium charlesii

The major exocellular glycopeptide (peptidophosphogalactomannan) produced by Penicillium charlesii first appears in the culture filtrate when the growth medium is nearly depleted of NH4+. The extent of incorporation of exogenously supplied radioactive precursors (d-[U-14C] glucose, l-[U-14C]threonine and NaH232PO4) into peptidophosphogalactomannan suggests that approximately 20% of the total quantity of peptidophosphogalactomannan is assembled from constituents taken from the growth medium before NH4+ starvation and that the remainder is assembled from constituents in the medium during NH4+ starvation. In the absence of NH4+, an increase in dry weight continues until the medium is depleted of glucose. However, peptidophosphogalactomannan accumulation proceeds until after glucose is depleted and growth is halted. These data suggest that peptidophosphogalactomannan is a product of cellular turnover.

The metabolism of tartaric acid by Penicillium charlesii

Abstract It was found that Penicillium charlesii metabolized 60–80% of all carbon atoms in dl -tartaric acid to carbon dioxide by 19 days after inoculation into a growth medium containing 32 m m l -tartaric acid, 278 m m d -glucose, and a mineral salts solution. A large fraction (80–90%) of the 14 C-labeled substances derived from tartrate, present in the mycelia after a 19-day growth period, was extracted into trichloracetic acid. Less than 1% of the labeled tartaric acid taken up by the organism was incorporated into protein. It was concluded that tartaric acid was not metabolized via a tartaric acid dehydratase-catalyzed reaction because aspartic acid derived from dl -tartrate-2,3- 14 C had slightly more than twice the specific activity of aspartic acid derived from dl -tartrate-1,4- 14 C. No tartrate dehydratase could be detected in any of the enzyme extracts. However, the organism contained enzymes capable of converting l -tartrate to glyceric acid, while an NAD-specific l -tartrate oxidoreductase and an NADPH-specific hydroxypyruvate oxidoreductase activity were demonstrated.

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol ☆

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with Km values of the order of 10(-4) M. A Km value of 60 mM was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(-)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(-)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.