Control Of Enzyme Synthesis Research Articles

Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress.

Immunolocalisation studies on six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2, in synovia from patients with osteo- and rheumatoid arthritis.

To assess the likely importance of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the arthritic process. Synovial samples from seven joints with rheumatoid arthritis and three osteoarthritic joints were analysed by indirect immunofluorescence microscopy. Using specific human antisera, we documented the frequencies and distributions of collagenase, stromelysins 1 and 2, matrilysin, gelatinases A and B, TIMP-1, and TIMP-2. Stromelysin 1 was found in all synovia, bound to extracellular matrix, within cells, or both, indicating stromelysin synthesis. Matrilysin was present in only one active inflammatory synovium, and focal synthesis of collagenase and gelatinase A was seen in four synovia. Stromelysin 2 and TIMP-2 were not observed, but TIMP-1 synthesis was seen in five synovia, and in two active synovia the distribution of TIMP-1 positive cells was more widespread than that of MMPs. The presence of stromelysin 1 in all synovia clearly implicates this enzyme in joint damage. Collagenase, gelatinase A and matrilysin may also have a role in rheumatoid arthritis, but are not significant in osteoarthritis. However, marked regional variations were found in the synthesis of these MMPs, indicating not only that these diseases are episodic but that control of enzyme synthesis is focal. Only TIMP-1 may be considered an inhibitory factor.

Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae. Evidence for a regulatory role of gene LYS14.

Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2-aminoadipate semialdehyde, an intermediate of this pathway. This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis. Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs. The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level. The synthesis of saccharopine dehydrogenase (glutamate-forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism. The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2-aminoadipate semialdehyde.

Control of enzyme synthesis in the oxalurate catabolic pathway of Streptococcus faecalis ATCC 11700: evidence for the existence of a third carbamate kinase.

Streptococcus faecalis ATCC 11700 uses oxalurate as a sole energy source for growth. An oxamate carbamoyltransferase and a carbamate kinase, both induced by oxalurate, are involved in this process. The oxalurate-induced kinase is specific for the pathway. Its properties are different from those of the previously characterized agmatine and arginine-induced kinases. Glucose, but not arginine, nor agmatine, two other energy sources, represses the oxalurate pathway. In contrast, oxalurate was found to be at least as effective as glucose in repressing the arginine deiminase pathway in arginine grown cells, or the agmatine deiminase pathway during growth on agmatine.

Control of enzyme synthesis in the arginine deiminase pathway of Streptococcus faecalis.

The formation of the arginine deiminase pathway enzymes in Streptococcus faecalis ATCC 11700 was investigated. The addition of arginine to growing cells resulted in the coinduction of arginine diminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3). Growth on glucose-arginine or on glucose-fumarate-arginine produced a decrease in the specific activity of the arginine fermentation system. Aeration had a weak repressing effect on the arginine deiminase pathway enzymes in cells growing on arginine as the only added substrate. By contrast, depending on the growth phase, a marked repression of the pathway by oxygen was observed in cells growing on glucose-arginine. We hypothesize that, in S. faecalis, the ATP pool is an important signal in the regulation of the arginine deiminase pathway. Mutants unable to utilize arginine as an energy source, isolated from the wild type, exhibited four distinct phenotypes. In group I the three enzymes of the arginine deiminase pathway were present and probably affected in the arginine uptake system. Group II mutants had no detectable arginine deiminase, whereas group III mutants had low levels of ornithine carbamoyltransferase. Group IV mutants were defective for all three enzymes of the pathway.

Modulation of lysosomal enzyme levels in cultured cells: Effects of alterations in cell density, balanced growth, and endocytosis

Abstract Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β- N -acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.

Evidence for preformed mRNA in the induction of TMP synthetase in Tetrahymena pyriformis

The induction of thymidylate (TMP) synthetase in Tetrahymena pyriformis occurred following a shift from complex medium to defined medium, or following addition of cyclic guanosine-3′,5′-monophosphate (cGMP) to complex medium. Inhibitors of macromolecular synthesis were used to probe the role of culture conditions in the enzyme induction. In defined medium actinomycin D minimally decreased the induction of enzyme activity, implying that control of enzyme synthesis is not exerted at the transcriptional level. Cycloheximide markedly inhibited the induction of TMP synthetase by defined medium, suggesting the requirement for protein synthesis in the induction process. In contrast, in complex medium where the activity of the synthetase was low (spec. act. <0.01), paradoxically, cycloheximide induced synthesis of the enzyme. This paradoxical effect was specific for cycloheximide, since neither puromycin nor pactamycin induced the enzyme. Further, the latter two both inhibited the inducing effect of cycloheximide by 60% and 81%, respectively. Actinomycin D only slightly (10%) lowered the paradoxical elevation in enzyme activity by cycloheximide. A possible utilization of preformed mRNA may underlie the basis of the increase in TMP synthetase specific activity in Tetrahymena pyriformis.

Control of enzyme synthesis and stability during sporulation in Saccharomyces cerevisiae

Studies were undertaken to understand the control of synthesis, stability and modification of UDP galactose epimerase and DNA-dependent RNA polymerase during sporulation of Saccharomyces cerevisiae. When a pre-induced culture of an inducible strain (wild type) is transferred to sporulation medium, the epimerase is inactivated to an undetectable level within 16 hours. Surprisingly, the addition of cycloheximide, a protein synthesis inhibitor, during sporulation stabilizes the epimerase activity. However, in a constitutive strain, the epimerase continues to be synthesized de novo during sporulation. Since the enzyme is synthesized during both vegatative growth and sporulation constitutively, the controls for synthesis of epimerase must be similar under these physiologically different conditions. After chromatography on DEAE Sephadex, there is no change observed in the elution patterns of RNA polymerase forms extracted from acetate growth vegetative cells, sporulating cells or from mature asci ; in all cases RNA polymerase consists of three forms, Ib, II and III. However, single spore suspension obtained from asci by treatment with zymolase contains a new form with chromatographic properties similar to those of form Ia. Our data suggests that form Ia may be a modification product of from Ib.

Control of enzyme synthesis by steroid hormones

Control of enzyme synthesis by steroid hormones

The effect of 8-hydroxyquinoline on enzyme synthesis in the fission yeast Schizosaccharomyces pombe.

The effect of 8-hydroxyquinoline, a rapid inhibitor of RNA synthesis, was followed on the activity of a number of enzymes in cultures of the fission yeast Schizosaccharomyces pombe. Two types of effect were found. In the first the activity continued to rise for a period and then remained constant. This occurred with alkaline phosphatase, basal and derepressed acid phosphatase, hexokinase, and derepressed sucrase and maltase at low cell density. It is consistent with control being exercised by an unstable mRNA or by an unstable stimulator of translocation. In the second the activity increased above the control values for several hours. This occurred with basal sucrase and maltase, and suggests a stable mRNA and an unstable inhibitor of translation. The extent of 'superproduction' of sucrase varied with cell density and with growth medium and this may be due to differences in the degree of translational inhibition. The possiblilty of a stable mRNA has interesting implications for the control of enzyme synthesis through the cell cycle.

Regulation in the foláte pathway of Escherichia coli

In Escherichia coli the great majority of one-carbon units enter the folate pathway as N 5 ,N 10 -methylene-THF. This compound is utilized directly in the biosynthesis of thymidylate, converted to N 5 -methyl-THF for use in methionine biosynthesis, and to N 10 -formyl-THF, which provides carbons 2 and 8 of purines and the formyl group of f-met-tRNA. Since the proportions utilized for different end-products can vary widely with changing conditions of growth, the distribution of N 5 ,N 10 -methylene-THF must be regulated in some way. We have studied the regulation of the conversion of N 5 ,N 10 -methylene-THF to N 10 -formyl-THF which is catalyzed by N 5 ,N 10 -methylene-THF dehydrogenase (MTDH) which converts N 5 ,N 10 -methylene-THF to N 5 ,N 10 -methenyl-THF, and N 5 ,N 10 -methenyl-THF cyclohydrolase (MTC), which converts N 5 ,N 10 -methenyl-THF to N 10 -formyl-THF. We concluded that the regulation could not be by control of enzyme synthesis since the specific activity of both enzymes increased with decreasing specific growth rate. N 10 -formyl-THF was found to be a noncompetitive, allosteric inhibitor of MTDH, and a competitive inhibitor of MTC. The value of K i for both inhibitions was about 5 × 10 −5 m . The intracellular concentration of total folates is in the range of 2 to 5 × 10 −4 m in E. coli ; thus the concentration of N 10 -formyl-THF will be such that significant inhibition will occur. The location of N 10 -formyl-THF in the folate pathway is such that its concentration can increase as its rate of utilization in purine biosynthesis decreases, as would be expected if the inhibition were to have regulatory significance; however, we do not yet have experimental proof that its concentration does change in the expected manner. Solutions have been calculated for the operation of a model folate pathway as it supplies the pathways of purine, deoxypyrimidine and methionine biosynthesis in a growing cell. The results of these calculations showed that inhibition of MTDH by N 10 -formyl-THF is advantageous to the cell, in that it permits a higher specific growth rate than if the inhibition did not occur; and that it can be essential, insofar as circumstances exist in which a steady state is impossible without the inhibition. However, it was also shown that inhibition of MTDH by N 10 -formyl-THF alone was insufficient to regulate the distribution of N 5 ,N 10 -methylene-THF when de novo purine biosynthesis is reduced by the addition of exogenous purines. We concluded that N 10 -formyl-THF inhibition of MTDH is a significant element in the regulation of MTDH, but that other mechanisms of regulation exist which we have yet to identify.

Cross pathway regulation of tyrosine and histidine synthesis in Bacillus subtilis: Biochemical, genetic, and transfer RNA studies

Abstract A large number of mutants resistant to the histidine analog, 1,2,4-triazole-3-alanine or the tyrosine analogs, d -tyrosine and fluorotyrosine were isolated in the wild type or bradytrophic strains of Bacillus subtilis . Most of these mutants had elevated levels of enzymes in both the tyrosine and histidine biosynthetic pathways. These mutants could be divided into six different groups based on the levels of these enzymes in cells grown on minimal medium and on medium supplemented with tyrosine or histidine. The gene or genes conferring resistance to these analogs mapped near the purB locus by DNA-mediated transformation. The activating enzymes and chromatographic patterns of tRNA for histidine and tyrosine were compared in preparations from wild type cells and several mutant classes, but there were no detectable differences in these components. The possibility that the common element in the control of enzyme synthesis in the tyrosine and histidine pathways is an aporepressor is discussed.

Corticosteroid Receptors and the Control of Enzyme Synthesis in Rat Liver

Corticosteroid Receptors and the Control of Enzyme Synthesis in Rat Liver

Influence of environment on the control of enzyme synthesis

Influence of environment on the control of enzyme synthesis

Influence of environment on the control of enzyme synthesis

Influence of environment on the control of enzyme synthesis

Control of enzyme synthesis in early sea urchin development: Aryl sulfatase activity in normal and hybrid embryos

Abstract Aryl sulfatase activity begins to increase at the mesenchyme blastula stage and continues to increase through the pluteus stage. Total activity increases 30-fold in Strongylocentrotus purpuratus embryos and 10-fold in Allocentrotus fragilis embryos. The bulk of new sulfatase activity is localized on or near the cell surface; thus, unlike the unfertilized egg or early cleavage stages, up to 75% of the sulfatase activity of larvae can be extracted with isotonic Na:K, and intact larvae can hydrolyze exogenous substrate. Mixing experiments do not reveal any soluble activators, but do indicate the presence of a heat-stable, dialyzable inhibitor in eggs and embryos. Developmental changes in this inhibitor, however, are not sufficient to account for the observed change in enzyme activity. Studies with viable hybrids between S. purpuratus and A. fragilis strongly suggest that increased aryl sulfatase activity is under nuclear control. Thus, total activity is intermediate to that of the two parents in both reciprocal crosses. Gel electrophoresis of the sulfatase in the hybrid embryos reveals a band of intermediate mobility to that of the two parents. Anomalously, however, there are no electrophoretic bands corresponding to the parental enzymes in the hybrid embryos. Although nuclear control is implied, inhibition of 85% of the protein synthesis by puromycin does not immediately arrest the increase in sulfatase activity. This suggests some sort of activation process which is independent of protein synthesis. Several models involving modification of protein after its synthesis are proposed to explain the lack of immediate effect of puromycin and the absence of the parental bands in the hybrid embryos. Although less probable, it is also proposed that the increase in sulfatase activity may result from the modification of a pre-existing protein by a factor or factors whose synthesis is under genetic control during development.

Control of enzyme synthesis by cellular interaction during development of the cellular slime mold Dictyostelium discoideum

Abstract The cellular slime mold Dictyostelium discoideum exists during part of its life cycle as a population of separate ameboid cells without obvious cellular interactions and at other times as an organized multicellular assembly of closely interacting cells capable of developing into either stalk cells or spores. The influence of cell interactions on this development was studied by measuring the accumulation of the enzyme UDP-glucose pyrophosphorylase, a pivotal enzyme needed for polysaccharide synthesis, during development. When partially developed cell aggregates were disaggregated to single cells and replated on a solid substratum, they reaggregated, rapidly recapitulated their previous morphological development, and then completed fruit construction with approximately normal timing. During this time they also repeated the program of enzyme accumulation forming an additional 300 units of UDP-glucose pyrophosphorylase activity regardless of the amount accumulated before disaggregation. In undisturbed developing cells the presence of actinomycin D did not affect pyrophosphorylase accumulation for several hours after its administration, indicating the prior accumulation of stable messenger RNA. After disaggregation, however, the presence of actinomycin inhibited any further increase in pyrophosphorylase activity, reaggregating cells being dependent on further RNA synthesis for the additional accumulation of this enzyme. This effect of disaggregation was not due to cell damage or to non-specific loss of RNA, as the rate of incorporation of [ 35 S]methionine into trichloroacetic acid-insoluble protein was the same in reaggregating cells as before disaggregation and was not affected for several hours by the presence of actinomycin. Disaggregated cells which were prevented from reforming normal cell contacts by EDTA formed little further enzyme and cells whose reaggregation was completely prevented by spatial separation formed none. The results indicate the need for cellular interaction to maintain the synthesis of this developmental enzyme even if the messenger RNA for the enzyme has already been formed.

The Two Operons of the Histidine Utilization System in Salmonella typhimurium

Abstract We have constructed an F factor bearing the genes (hut) responsible for the synthesis of the histidine-degrading enzymes in Salmonella typhimurium. This was accomplished by transducing the genes from the chromosome onto an F factor from Escherichia coli bearing the gal-bio region, which brackets the hut genes but does not include them. Recovery of this nonhomologous transduction was made possible by deleting the homologous hut region from the chromosome. Studies of the control of enzyme synthesis in heterozygous merodiploids showed that the hut region is composed of two operons. One operon includes the structural genes for histidase and urocanase, the first two enzymes of the pathway, and a promoter-operator complex controlling their expression in cis. The second operon includes the structural genes for 4-imidazolone-5-propionate amidohydrolase and N-formimino-l-glutamate formiminohydrolase, the last two enzymes of the pathway, and a promoter gene controlling their expression in cis. Located between these two operons is a regulatory gene, whose product presumably represses both operons in trans. The target site of catabolite repression of histidase and urocanase is the promoter of their operon, as is the case in the lac operon of E. coli.

Metabolic Regulation in Glucose-Limited Chemostat Cultures of Escherichia coli

Glucose-limited chemostat cultures of Escherichia coli, growing at dilution rates above 0.3/hr, continue to grow at the restricted rate after removal of glucose restriction. In a glycogenless strain, the specific rates of increase of mass, protein, and ribonucleic acid (RNA) were equal before and after supplementation with 0.05% glucose and did not increase detectably until after 30 to 60 min. The unrestricted specific growth rate was reached after two to three doublings of cell mass. Supplementation with glucose plus 20 amino acids, but not with glucose plus vitamins or ribosides, produced an immediate increase in the specific rates of mass and RNA synthesis followed by an increase in the specific rate of protein synthesis. In a wild-type strain, synthesis of protein and RNA continued at the restricted rate after glucose supplementation, but the specific rate of increase of mass immediately increased due to rapid synthesis of glycogen. At dilution rates less than 0.3/hr, the specific rates of increase of mass, protein, and RNA increased immediately after supplementation with glucose, but did not immediately attain the unrestricted growth. The results at dilution rates greater than 0.3/hr are interpreted to mean that the regulation of a number of enzymatic reactions is entirely through control of enzyme synthesis, without modulation of enzyme function. The levels of such enzymes are controlled so that operation with zero-order kinetics precisely meets the demands for balanced growth. It was shown that glutamic dehydrogenase and glutamic-oxalacetic transaminase are regulated in this manner.

Food-stimulated synthesis of intestinal proteolytic enzymes in the cockroach Leucophaea maderae

Abstract In adult Leucophaea males and females high proteolytic activity is primarily found in the posterior region of the midgut lumen. Only feeding on rat food, casein, fibrin, glutenin, and elastin stimulated protease activity; several other proteins, casein hydrolysates, amino acids, some polyamino acids, and starch did not cause increased enzyme activity. A proportionality was found between the quantity of certain proteins passed into the midgut and the enzyme activity in the midgut. Since the rate of the food release from the crop into the midgut is higher the more food is eaten, the level of enzyme activity is positively related to the amount of food consumed during one meal. The first increase in enzyme activity was observed about 12 to 14 hr after feeding; the enzyme level stayed high for approximately 1·5 days and then decreased again. After injection of 14 C-leucine into fed or starving animals, fractionation on Sephadex G-150 revealed that only the enzyme fractions of fed animals were appreciably labelled; little label was found in the enzyme fractions from starving animals. This is support for the hypothesis that proteinaceous foods stimulate protease synthesis rather than activate a precursor. No control of enzyme synthesis appears to be exerted by hormones from the brain (neurosecretory cells), corpora cardiaca, or corpora allata since removal of these organs before or after feeding had no effect on protease production in the midgut. However, severance of the innervation of the proventricular valve caused a reduction of enzyme synthesis because food passage was impaired after this operation. It is concluded that stimulation of protease synthesis in the midgut is solely secretagogue. A powerful inhibitor for the cockroach protease in found in the anterior midgut and the caeca. It is apparently produced in the caeca at a high rate. Its characteristics are similar to those of soy bean trypsin inhibitor; it is heat-stable, non-dialysable, can be digested by pronase and the animal's own proteases, and is precipitable by ammonium sulphate. It is thus likely that the inhibitor is a heat-stable protein.