Simultaneous induction of three catabolic enzymes inEscherichia coli

During a simultaneous induction of three enzymes which are subject to catabolite repression (β-galactosidase, tryptophanase and amylomaltase, or β-galactosidase, tryptophanase and D-serine deaminase) in a batch culture, the rates of synthesis of β-galactosidase and tryptophanase decreases, while the rates of synthesis of amylomaltase and D-serine deaminase remain unaffected. The addition of cAMP brings about a considerable increase of the rate of synthesis of D-serine deaminase and a partial synthesis rate increase of β-galactosidase while the synthesis rate of tryptophanase remains lowered and the synthesis rate of amylomaltase remains unaffected. In a continuous culture β-galactosidase, tryptophanase andD-serine deaminase are synthesized simultaneously at a maximum rate without mutual influence. The addition of cAMP increases the rate of synthesis of all three enzymes.     

Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Regulation of nitrogen catabolic enzymes in Streptomyces clavuligerus

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligerus in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.     

Laboratory evolution of catabolic enzymes and pathways

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.     

Genetic variation in the activity of the histidine catabolic enzymes between inbred strains of mice: A structural locus for a cytosol histidine aminotransferase isozyme (Hat-1)

Variation in activity of the main histidine catabolic enzymes (histidase, urocanase, and aminotransferase) has been surveyed using inbred strains of mice (C57BL, DBA, Peru, SM, and SWR). Some variation was found in the activity of all enzymes, but only in the case of cytosolic histidine aminotransferase was it greater than twofold (SM 3.3-fold greater than C57BL). The divergent strains for the activity of this enzyme were crossed and the F ₁ 's were backcrossed; the segregation analysis indicated a single locus with additively acting alleles (designated Hat-1: a allele SM, b allele C57BL). Cytosolic histidine aminotransferase differed in heat stability between SM and C57BL, indicating that Hat-1 is a structural locus. The conflict in the biochemical literature (Morris et al., 1973; Noguchi et al., 1976a, b) over the number and subcellular distribution of the histidine aminotransferase isozymes is partly resolved by the acquisition of a variant at the Hat-1 locus. Hat-1 affects the cytosolic form but not the mitochondrial form of the enzyme. Purification and analysis of the isozymes of histidine aminotransferase from livers of C57BL and SM mice will further clarify the situation.     

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.     

NADP+ catabolic enzymes in differentiating rabbit erythroid cells

NADP-glycohydrolase and NADP-pyrophosphates activities were examined during the rabbit erythroid cell differentiation. The former is high in erythroblast lysates, especially in the erythroblast nuclei. As erythroid cell maturation proceeds, the activity of NADP-glycohydrolase decreases. At the first step (erythroblast-reticulocyte transformation), this activity falls down more than by 20 times, whereas at the second step (reticulocyte-erythrocyte transformation) it decreases no more than twice. NADP-glycohydrolase is associated with the stroma of erythroid cells throughout their maturation, being bound with the nucleus in erythroblasts. NADP-pyrophosphatase activity has been detected in reticulocytes and mature cells only. The role of NADP- and NAD-glycohydrolases for characterization of the intracellular metabolic pools is discussed.     

Regulation of the purine catabolic enzymes in Neurospora crassa.

Neurospora crassa can utilize purines and their metabolic products as a nitrogen source. Regulation of the five enzymes required for uric acid metabolism was studied. The first three enzymes of this catabolic pathway are controlled in a complex manner that involves both induction and repression. Both uricase and allantoicase were induced by uric acid while allantoinase was induced by either uric acid or allantoin. Synthesis of all three of these enzymes was repressed by the end product, ammonia. The ure-2 mutant, which is urease deficient and cannot derive ammonia from purines, shows a hyperinducibility of these same three enzymes. The last two enzymes of the pathway, ureidoglycollase and urease, were found to be constitutive.     

Kinetics growth-plate dependent formation of catabolic enzymes in escherichia coli

Summary Experimental data on the production of several inducible catabolic enzymes in Escherichia coli in conditions of batch and continuous cultivation were used for the evaluation of the dependence of specific rate of enzyme synthesis on the RNA content and specific growth rate. The results show that the Jacob-Monod theory for enzyme synthesis cannot be extended to conditions of limited growth.