Terry S. Woodin

Evidence for three isozymes of chorismate mutase in alfalfa

Abstract Three forms of chorismate mutase (CM1, CM2, CM3) have been found in alfalfa plants. The isozymes are separable by DEAE-cellulose fractionation and gel electrophoresis. They are distinguishable by different molecular weights (46 000, 58 000 and 69 000, respectively), different activation energies (11 300, 20 800 and 13 500 cal/mole, respectively) and differing sensitivity to metabolic inhibitors, CM1 and CM3 are inhibited by phenylalanine and tyrosine and activated by tryptohphan, CM1 and CM2 are inhibited by caffeic and chlorogenic acid. CM3 is unaffected by these dihydroxycinnamic compounds, but is activated by 3,4-dimethoxycinnamic acid and inhibited by ferulic acid, two compounds which have no effect on CM1 and CM2. All isozymes are inhibited by p-coumaric acid. All three forms are present in all plant parts of the mature alfalfa and in green as well as etiolated seedling.

Drosophila melanogaster fatty acid synthetase: Characteristics and effect of protease inhibitors

Abstract Fatty acid synthetase (FAS) from Drosophila melanogaster was purified by DEAE-cellulose and Sepharose CL-6B chromatography. Inclusion of protease inhibitors in all steps dramatically increased the specific activity of the FAS preparation (to an average value of 4500 U/mg protein, the highest value reported for any animal FAS). The relative molecular weight of the native enzyme was determined by gel filtration and found to be 480,000. SDS gel electrophoresis gave a subunit relative molecular weight of 226,000, indicating that D. melanogaster FAS, like other animal FASs, is a dimer. Acetyl-CoA was the most efficient primer with propionyl-CoA also supporting FAS activity. Neither hexanoyl-CoA butyryl-CoA, isobutyryl-CoA nor isovaleryl-CoA served as efficient primers. D. melanogaster FAS showed an absolute requirement for malonyl-CoA and no activity was observed when methylmalonyl-CoA replaced malonyl-CoA. However, in the presence of both elongating substrates, D. melanogaster FAS synthesized methyl branched fatty acids. Methylmalonyl-CoA appears to behave as a competitive inhibitor in the presence of malonyl-CoA

Improved assay for phenylpyruvic acid

An improved method for the assay of phenylpyruvate using 1 M borate/2 M phosphate (pH 6.5) to develop the phenylpyruvate enolborate complex is described. A reinvestigation of the extinction coefficient of phenylpyruvate in various normalities of NaOH is also reported. Both methods are compared for the assay of chorismate mutase.

Chorismate mutase isozyme patterns in three fungi

Abstract As judged by results of gel electrophoresis and effector sensitivity, the number of chorismate mutase isozymes differs in extracts of the three fungi investigated. Neurospora crassa contains only one form of chorismate mutase designated CM 1 . It is inhibited by phenylalanine, tyrosine and caffiec acid, activated by tryptophan and unaffected by ferulic and 3,4-dimethoxycinnamic acid. Peniccilium chrysogenum contains two mutases; a CM 1 similar in electrophoretic mobility and effector sensitivity to that of N. crassa and CM 3 which is inhibited by phenylalanine and tyrosine, activated by tryptophan and 3,4-dimethoxycinnamic acid but unaffected by caffeic acid. Penicillium duponti a thermophilic fungus, contains three forms of the mutase; a CM 1 , and a CM 3 similar in electrophilic mobility and effector sensitivity to those of P. chrysogenum and CM 2 , which is inhibited by caffeic acid, slightly activated by tryptophan and unaffected by phenylalanine, tyrosine or ferulic acid.

Chemical state of magnesium in plasma

Abstract All the magnesium in ovine and bovine plasma behaves as a low molecular weight cationic complex. No plasma magnesium is associated with protein.

Anomalous behavior of magnesium ion on Sephadex G-10

Abstract Magnesium salts were chromatographed on Sephadex G-10 and Bio-Gel P-2 with pH 7.0, 0.010 M potassium phosphate as the principal eluting buffer. The elution volume and chromatographic profile of the magnesium ion was affected by a variety of factors including the nature of the anionic counter-ion, the ionic strength of the salt solution applied, and the nature and concentration of co-applied anions and cations. The most striking feature of the behavior of magnesium ion during chromatography on Sephadex G-10 is that under certain conditions it disproportionates into two peaks, one with an elution volume typical of a small ion with complete access to the gel and the other typical of a larger ion.

Sulfate permease ofPenicillium duponti

Abstract The thermophilic fungus Penicillium duponti has an active sulfate permease system derepressible by growth in cysteic acid or by sulfur starvation. The system is sensitive to ionic strength and is optimal at 45°C, pH 6.5. It is saturable with an apparent K m for sulfate of 55 μ M . It is activated strongly by divalent cations and inhibited by inhibitors of mitochondrial ATP production and the group VI oxyanions.

Effect of temperature on sulfate transport in two Penicillia

Abstract We studied the effects of temperature on the sulfate permease of Penicillium chrysogenum (PC) (a mesophile with a growth temperature range of 4-35°C) and Penicillium duponti (PD) (a thermophile with a growth temperature range of 27-58°C). Arrhenius plots of sulfate permease activity from mycelia grown at 50°C (PD), 30°C (PD and PC) or 8°C (PC) indicate that at temperatures below the transition point there is little difference in the activation energy of sulfate permease in mycelia from PD grown at 50°C or 30°C or PC grown at 30°C or 8°C; however, the temperature of the transition point for the permease from each set of mycelia assayed reflects the optimum growth temperature of the fungal source. Transitions occur at 15 °C for mycelia from PC and 35 ° C for PD mycelia. Kinetic measurements indicate that the K m of sulfate permease in PC cells grown at a variety of temperatures is essentially the same at various temperatures. As an example, the K m of 8°C or 30°C grown PC is about 55 μ M at 25°C and 45 μ M at 8°C. V max measurements reflect growth conditions such as temperature and growth stage. p]Lipid composition of the mycelia dramatically reflect growth temperatures. Double bond index values vary from 1.94 for PC grown at 8°C to 0.81 for PD grown at 50°C. The percentage of total fatty acid represented by linolenic acid varies from 45% in 8°C grown PC to 4.2% or less in 30°C grown PC. No linolenic is found in mycelia from PD.

Distribution of plasma magnesium in various vertebrates

Plasma magnesium in at least five mammalian species (humans, rat, dog, sheep, cattle) is in the form of a complex, separable from ionic magnesium and plasma protein by size exclusion chromatography on Sephadex G-10. Plasma magnesium in three non-mammalian vertebrates (toads, trout, chicken) behaves similarly to ionic magnesium or as a very small magnesium complex on Sephadex G-10.

Comparative Arrhenius plots of enzyme activity from penicillium

Comparison of the Arrhenius plots of three enzymes, formyltetrahydrofolate synthetase, glutathione reductase (GSSGR) and chorismate mutase (CM) from a thermophilic (Penicillium duponti) and a mesophilic (Penicillium chrysogenum) fungus reveals a fairly consistent pattern. In general, those enzymes extracted from mesophiles had lower activation energies than similar enzymes extracted from thermophiles. One enzyme studied, mesophilic glutathione reductase, exhibited a break in its Arrhenius plot. The allosteric enzyme studied showed slightly different sensitivities in the thermophilic versus the mesophilic extracts.