Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, α-D-galactose-1-phosphate:UDP glucose uridylyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increased cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.     

Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells.

The specific activities of three murein hydrolases, carboxypeptidase I, carboxypeptidase II, and amidase were studied with respect to cell division in toluene-treated cells of Escherichia coli K-12. Carboxypeptidase I and amidase activities were constant throughout the division cycle in cells of D11/lac+pro+. Detectable carboxypeptidase II activity varied and was highest at the time of division by a factor of three. Carboxypeptidase II specific activity was also correlated with cell division in BUG 6, a temperature-sensitive mutant (J.N Reeve, D.J. Groves, and D.J. Clark, 1970). Fifteen minutes after shifting BUG 6 from 42 C (nondividing conditions) to 32 C (dividing conditions), there was a rapid resumption of cell division, accompanied by a 10-fold increase in the specific activity of carboxypeptidase II. These results demonstrate a correlation between detectable carboxypeptidase II activity and cell division as reflected by activity in toluene-treated cells. The subcellular location of carboxypeptidase II, a soluble enzyme was found to be periplasmic since it was released by tris(hydroxymethyl)-aminomethane-ethylenediaminetetraacetate treatment and osmotic shock, two methods known to release periplasmic enzymes.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae

Conditions were established for the assay of three nucleolytic enzymes: a Mg2+-independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5'-exonuclease in Saccharomyces cerevisiae cell extracts. The changes in the activities of these enzymes were determined throughout the life cycle of the organism. As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5'-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased. During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation. However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels. The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells. The Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease showed little change during germination of the ascospores. The high specific activity of the Mg2+-independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions.     

Cultured human fibroblast enzymes. III. Enzyme activity in a triploid strain]

Complex investigation of 5 enzymes was carried out in a cell strain with triploidy 69, XXY, derived from a human spontaneous abortus. The activity of 3 enzymes (acid phosphatase, lactate and malate dehydrogenases) in triploid cells proved to be significantly increased as compared to those of 3 diploid strains, whereas the activity of alkaline phosphatase was decreased. The activity of glutamate-oxalacetate transaminase did not change. The absence of the pronounced genetic dose effect and different alteration of the activities of the enzymes studied may be considered as an expression of a disbalance of enzymes in cytogenetically defective cells.     

植被修复对重度盐碱地土壤酶活性和酶反应热力学特征的影响

由于环境变化和人为干扰,松嫩平原盐碱化土地面积不断增加,盐碱化程度也在不断加剧。本试验以松嫩平原西部盐碱化土壤为对象,研究了自然植被、羊草、燕麦和苜蓿修复对重度盐碱化土壤2种氧化还原酶(过氧化氢酶、多酚氧化酶)和3种水解酶(碱性磷酸酶、蔗糖酶、脲酶)活性和酶反应热力学特征的影响。结果表明: 随着温度的升高,5种土壤酶活性、活化自由能(ΔG)都逐渐升高,在40 ℃(或45 ℃)时达到最高,而土壤酶的活化焓(ΔH)和活化熵(ΔS)无显著变化;温度每增加10 ℃时的温度系数(Q₁₀)变化较小,范围为1.05～1.36。与裸地相比,过氧化氢酶活性在自然植被和羊草修复地升高,而在燕麦和苜蓿修复地降低,ΔG和酶活性的变化规律则相反,而ΔH、ΔS在羊草、燕麦修复地升高,在自然植被和苜蓿修复地降低;多酚氧化酶活性在各修复地都降低或不变,ΔH、ΔS在自然植被和羊草修复地降低,而在燕麦和苜蓿修复地不变,ΔG在各修复样地都表现为在40 ℃时升高,在其他温度下均降低或不变。在各修复地,3种水解酶活性均升高,酶的ΔG降低或不变,而ΔH和ΔS则差异较大。土壤酶活性及其热力学特征对温度变化和植被修复的响应存在较大差异。研究结果可为重度盐碱化土壤的修复提供理论依据。     

Changes in fungi with age. II. Respiration and respiratory enzymes of Rhizoctonia solani and Sclerotium bataticola

Van Etten, James L. (University of Illinois, Urbana), H. Peter Molitoris, and David Gottlieb. Changes in fungi with age. II. Respiration and respiratory enzymes of Rhizoctonia solani and Sclerotium bataticola. J. Bacteriol. 91:169-175. 1966.-The rate of respiration of Rhizoctonia solani and Sclerotium bataticola decreased with age. This decrease in respiratory rate might be produced by a decrease in the specific activity of one or more enzymes involved in carbohydrate metabolism. Specific activities in cell-free extracts were measured for most of the enzymes in the hexose monophosphate shunt, Embden-Meyerhof-Parnas pathway, tricarboxylic acid cycle, and terminal electron-transport system. In addition, glucose oxidase, isocitritase, and malic enzyme were measured. In R. solani, increases in activity with age occurred for hexokinase, alpha-glycerolphosphate dehydrogenase, malic dehydrogenase, and cytochrome oxidase. Decreases occurred for phosphohexokinase, aconitase, nicotinamide adenine dinucleotide-specific isocitric dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and at least one of the enzymes between 3-phosphoglycerate and pyruvate. In S. bataticola, increases in activity with age were observed for phosphohexokinase, pyruvic dehydrogenase, fumarase, malic dehydrogenase, and malic enzyme, whereas none of the enzymes decreased. The specific activities of the remaining enzymes did not change with age in either fungus.     

Enzymes of galactose metabolism in erythrocytes and liver of inbred strains of mice.

This study compares the activity of galactokinase, galactose-1-phosphate uridyltransferase, and UDPgalactose-4-epimerase, the important enzymes in the pathway of conversion of galactose to glucose in red cells and liver of five inbred strains of mice. In the red cells, galactokinase varied over a four-fold range of activity while the other enzymes varied about two-fold. The activity of each of the enzymes varied independently of the other so that red cells of each strain had a unique pattern for the three enzymes. The red cell activity pattern was not reflected in liver tissue which showed little interstrain variation for each of the three enzymes. The ratio of liver galactokinase to uridyltransferase and epimerase was very similar in all five strains. Oxidation in vivo of ¹⁴C galactose to ¹⁴CO₂ was examined in the two strains of mice with the widest divergence of red cell galactokinase activity and no difference was found in this parameter measuring the physiological disposition of the sugar. The wide variation of the red cell enzyme activity appears to have little metabolic consequence for the animal, the oxidation of the sugar reflecting the relative constancy of the liver enzyme activity.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.     

Control of the flux to arginine in Neurospora crassa: de-repression of the last three enzymes of the arginine pathway

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold.By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.     

Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis

Cells of Bacillus thuringiensis containing refractile spores autolyzed readily when suspended in buffer. The autolysate contained enzymes which lysed vegetative cell walls of the organism. Three enzymes were isolated from the autolysate, and each was purified approximately 30-fold. One enzyme, most active near pH 4.0, was found to be an N-acetylmuramidase. The other two enzymes exhibited pH optima at 8.5. One was stimulated by cobalt ions and the other was not. The cobalt-stimulated enzyme was shown to be an N-acetylmuramyl-l-alanine amidase. The cobalt insensitive enzyme exhibited both N-acetylmuramyl-l-alanine amidase and endopeptidase activity. The amidase activity may reflect incomplete separation of the cobalt-stimulated enzyme. The endopeptidase cleaved the peptide bond between l-alanine d-glutamic acid. A cell wall lytic endopeptidase with this specificity has not been previously reported. All three enzymes were extremely limited in the range of bacterial cell walls which they attacked. Except for cell walls of Micrococcus lysodeikticus, which were lysed by the muramidase, only cell walls of members of the genus Bacillus were attacked.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

The effect of beta-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ.

1. The rate of mammary-gland lipogenesis measured in vivo from 3H2O was suppressed after decreasing the milk demand by decreasing the number of pups from ten to two or three, as well as by giving diets containing lipid [Grigor & Warren (1980) Biochem. J. 188, 61-65]. 2. The specific activities of the lipogenic enzymes fatty acid synthase, glucose 6-phosphate dehydrogenase and 'malic' enzyme increased between 6- and 10-fold in the mammary gland and between 2- and 3-fold in the livers during the first 10 days of lactation. The increases in specific activity coupled with the doubling of liver mass which occurred during pregnancy and lactation resulted in considerable differences in total liver activities when compared with virgin animals. 3. Although consumption of a diet containing 20% peanut oil suppressed the activities of the three lipogenic enzymes in the livers, only the 'malic' enzyme was affected in the mammary glands. 4. In contrast, decreased milk demand did not affect the specific activities of any of the liver enzymes, whereas it resulted in suppression of all three lipogenic enzymes of the mammary glands. There was no effect on either the cytoplasmic malate dehydrogenase or the lactate dehydrogenase of the mammary gland. 5. In all the experiments performed, the activity of the fatty acid synthase correlated with the amount of material precipitated by the rabbit antibody raised against rat fatty acid synthase.     

Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes.

Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes.     

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1-2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.     

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1–2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.     

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).Abbreviations ABA abscisic acid - bp base pairs - DPA days post anthesis - HPI hours post imbibition - kb kilobase (pairs) - M _r relative molecular weight - S Svedberg unit (10^-13s)     

Regulation of skeletal muscle metabolism by enzyme binding.

The random diffusion mechanism is usually assumed in analyzing the energetics of specific pathways despite the findings that enzymes associate with each other and (or) with various membranous and contractile elements of the cell. Successive glycolytic enzymes have been shown to associate in the cytosol as enzyme complexes or bind to the thin filaments. Furthermore, the degree of glycolytic enzyme interactions have been shown to change with altered rates of carbon flux through the pathway. In particular, the proportions of aldolase, phosphofructokinase, and glyceraldehyde phosphate dehydrogenase bound to the contractile proteins have been found to increase with increased rates of glycolysis. In addition, decreasing pH and ionic strength are also associated with an increase in glycolytic enzyme interactions. The kinetics displayed by interacting enzymes generally serve to enhance their catalytic efficiencies. The associations of the glycolytic enzymes serve to enhance metabolite transfer rates, increase the local concentrations of intermediates, and provide for regulation of activity via effectors. Therefore these interactions provide an additional mechanism for regulating glycolytic flux in skeletal muscle.