Henry C. Reeves

Rapid isoelectric focusing in a vertical polyacrylamide minigel system

A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day.

NADP+-specific isocitrate dehydrogenase of Escherichia coli: I. Purification and characterization

Abstract The NADP + -dependent isocitric dehydrogenase of Escherichia coli E-26 was purified to electrophoretic homogeneity and partially characterized. The purification was carried out at room temperature and resulted in a 64% recovery of the enzyme from the crude cellular extract. The enzyme had a molecular weight of 80 000 as determined by gel filtration and sucrose density gradient centrifugation. The K m values for threo - d s -isocitrate, NADP + , Mg 2+ and Mn 2+ were 1.56 · 10 −5 , 3.7 · 10 −5 , 1.27 · 10 −4 , and 1.29 · 10 −5 M, respectively. The coenzyme analogs, thionicotinamide-NADP + and 3-acetylpyridine-NADP + , but not selenonicotinamide-NADP + , were able to replace NADP + in the reaction. The enzyme exhibited maximum stability at pH 6 and had a broad pH optimum from 7 to 9.

Growth of Escherichiacoli on fatty acids: Requirement for coenzyme a transferase activity

Abstract The ability of an Escherichia coli strain (E-26) to grow on butyrate or valerate as the sole source of carbon has previously been correlated with the glyoxylate-short chained fatty acid acyl-CoA ester condensing activities (1). In attempting to confirm and expand these results, several strains of Escherichia coli K12, of previously defined genotype and physiology (2) were employed. From one of them, constitutive for the enzymes of the glyoxylate shunt, a new mutant, V10, has been selected for its ability to grow on butyrate or valerate as the sole source of carbon. The growth of this strain on these fatty acids is rapid and is not preceded by any significant lag when transfered from a glucose medium. This physiological behavior is related to the presence of a constitutive level of an acyl-CoA: acetate transferase activity. This enzymatic reaction, not previously reported in Escherichia coli , is measurable by spectrophotometric assays and a radioisotopic microassay. A second mutant, derived from V10, is simultaneously deficient in the transferase activity and in the ability to grow on butyrate or valerate. The data presented suggest that the required transferase functions as an activating mechanism which permits the organism to utilize these fatty acids as a carbon source for growth.

NADP+-specific isocitrate dehydrogenase of Escherichia coli. II. Subunit structure.

Abstract The subunit structure and molecular weight of Escherichia coli NADP+-specific isocitrate dehydrogenase has been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate at pH 7.0 and in 8 M urea at pH 2.9. The results obtained indicate that the enzyme, which exists as a catalytically active dimer, is composed of two subunits each with a molecular weight of approximately 42 000. Cross-linking with dimethylsuberimidate, followed by electrophoresis in sodium dodecylsulfate gives, in addition to a band of mol. wt 43 000, a second protein band of approximately 83 000. A single NH2-terminal amino acid, methionine, was obtained following dansylation of the performic acid oxidized protein suggesting identity of the subunits. The isoelectric point of the E. coli isocitrate dehydrogenase was found to be pH 5.0.

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D5H3G7. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The Km of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 μM. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.

NADP-specific isocitrate dehydrogenase of Escherichia coli: IV. Purification by chromatography on Affi-Gel Blue

Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) from Escherichia coli. The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature. The preparation was judged to be homogeneous by non-denaturing electrophoresis at pH 7.5 and denaturing electrophoresis in the presence of sodium dodecyl sulfate. The subunit molecular weight of 53 000, determined by sodium dodecyl sulfate gel electrophoresis, is in reasonable agreement with the value of 46 900 estimated from the amino acid composition data.

Multiple Forms of Bacterial NADP-Specific Isocitrate Dehydrogenase

Electrophoretically distinct forms of nicotinamide adenine dinucleotide phosphate-specific isocritrate dehydrogenase have been observed in extracts of Escherichia coli grown under different culture conditions. In glucose-grown cells, two distinct bands of isocitrate dehydrogenase activity were observed on polyacrylamide gels and have been completely resolved by employing ion-exchange chromatography. These multiple forms of the enzyme have been studied and their possible metabolic role is discussed.

Escherichia coli isocitrate lyase: properties and comparisons

The glyoxylate cycle was first discovered during studies on bacteria and fungi with the ability to grow on acetate or ethanol as the sole carbon source. Isocitrate lyase, the first enzyme unique to the glyoxylate cycle, has been studied in numerous prokaryotic and eukaryotic organisms. However, information on this enzyme from Escherichia coli is limited. We have recently reported the purification and in vitro phosphorylation of this enzyme. In the present study we have examined and characterized a variety of inhibitors, the divalent cation requirement and the amino acid composition of E. coli isocitrate lyase and compared these results to those obtained with other organisms.

Invivo phosphorylation of isocitrate lyase from Escherichiacoli D5H3G7

This report describes the in vivo phosphorylation of isocitrate lyase and examines the possible consequences to the control of the Krebs cycle and glyoxylate bypass. NADP-specific isocitrate dehydrogenase from E. coli was the first bacterial protein whose enzymic activity was shown to be modulated by reversible phosphorylation. This enzyme has been thought to be solely responsible for the partitioning of isocitrate between the Krebs cycle and glyoxylate bypass. No studies to date have examined the possible role of isocitrate lyase in controlling this flux.

Propionate oxidation in Escherichia coli

An investigation was undertaken to determine the principal pathway of propionate oxidation in Escherichia coli E-26. Studies in a number of systems have established that propionate may be metabolized by several different pathways. Since these pathways may be differentiated by the pattern of14CO2 evolution from specifically labelled carbon atoms of propionate, a radiorespirometric experiment was performed in which propionate-adapted E. coli E-26 was used. Under these conditions, both the rate and final cumulative percentage of14CO2 was greatest for 1-14C-propionate, intermediate for 2-14C-propionate, and least for 3-14C-propionate. The observed labelling pattern is consistent with oxidation of propionate to acetate via lactate.