Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocysts. The enzyme activities are distinguishable by means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-l-lysine-chloromethyl ketone or l-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.     

Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.     

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic reorganization and signal transduction during estivation in the spadefoot toad

The maximal activities of 28 enzymes, representing multiple pathways of intermediary metabolism, were quantified in the brain, liver and skeletal muscle of spadefoot toads Scaphiopus couchii, comparing control toads with animals that had estivated for 2 months. Estivation-induced changes in brain enzyme activities were consistent with suppressed glycolysis and increased ketone body and amino acid catabolism. In liver, estivation resulted in reduced activities of eight enzymes representing carbohydrate, amino acid, ketone body and phosphagen metabolism, but the maximal activity of malic enzyme increased by 2.4-fold. Estivation led to a large-scale reorganization of skeletal muscle affecting most of the enzymes analyzed. Activities of enzymes of carbohydrate catabolism were generally elevated except for glycogen phosphorylase and hexokinase, whereas those of enzymes of fatty acid synthesis and ketone body metabolism were reduced. Increased glutamate dehydrogenase activities in both brain and muscle, as well as activities of other amino-acid-catabolizing enzymes in muscle, correlated with specific changes in the free amino acids pools in those tissues (reduced glutamine activity, increased glutamate, alanine and valine activities) that appear to be related to protein catabolism, for the purposes of elevating urea levels. The effects of estivation on signal transduction systems were also assessed. Total activities of protein kinases A and C (PKA and PKC) were largely unaltered in toad tissues during estivation (except for a 57% reduction in liver total PKC), but in seven organs there were strong reductions in the percentage of PKA present as the active catalytic subunit in estivating animals, and three contained a much lower percentage of membrane-bound active PKC during estivation. Activities of protein phosphatase types 1, 2A, 2B, and 2C were also frequently reduced during estivation. Overall, these results suggest that anuran estivation involves metabolic reorganization, including changing the maximal activities of key enzymes of intermediary metabolism as well as depressing the metabolic rate by suppressing signal transducing enzymes.     

马尾松人工林土壤有机层和矿质土壤层酶活性随雨旱季的变化

土壤酶活性随雨旱季的变化特征对于深入理解人工林土壤物质循环具有重要意义。2014年6月至2017年4月,采用原位土柱培养方法,研究了长江上游低山丘陵区的马尾松人工林土壤有机层和矿质土壤层转化酶、脲酶和酸性磷酸酶活性随关键时期(雨季初期;雨季中期;雨季末期;旱季初期;旱季末期)的变化特征。结果表明：土壤有机层和矿质土壤层酶活性随关键时期的变化显著,总体表现为雨季高于旱季。土壤有机层转化酶和脲酶活性呈现逐年降低的趋势;矿质土壤层酸性磷酸酶活性呈现逐年增加的趋势。土壤有机层转化酶和酸性磷酸酶活性显著高于矿质土壤层。土壤有机层三种酶活性年际变化幅度及随关键时期的变化幅度均高于矿质土壤层。偏最小二乘分析(PLS)表明,土壤含水量、微生物量和底物含量对土壤酶活性具有显著影响,但其影响大小强烈取决于土壤层次和酶的种类。可见,马尾松人工林土壤有机层酶活性对环境因子变化的响应更敏感,而且驱动土壤有机层和矿质土壤层酶活性动态的主导因子也存在差异。     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

Transition of metabolisms in living popular bark from growing to wintering stages and vice versa: changes in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities and in the levels of sugar phosphates

Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar (Populus gelrica). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins.     

Lipogenic enzymes in rat maternal adipose tissue in the perinatal period.

1. The specific activities of fatty acid synthetase, acetyl-CoA carboxylase and pyruvate dehydrogenase were measured in rat adipose-tissue extracts in pregnancy and lactation. Fatty acid synthetase specific activity correlates very closely with the rate of fatty acid synthesis, the enzyme specific activity decreasing after mid-pregnancy in a manner very similar to the rate of fatty acid synthesis. Acetyl-CoA carboxylase specific activity also decreases dramatically after mid-pregnancy. Initial pyruvate dehydrogenase specific activity shows a decrease between 2 days pre partum and 2 days post partum, but total enzyme activity shows no significant change in the same period. 2. Immunotitrations of fatty acid synthetase and pyruvate dehydrogenase activities were carried out; the titrations showed that the change in the fatty acid synthetase activity is due to a change in the enzyme amount; the amount of pyruvate dyhydrogenase does not change. Therefore the decrease in fatty acid biosynthesis in subcutaneous and parametrial adipose tissue in late pregnancy and early lactation is associated with a decrease in the amount of at least one of the enzymes involved in fatty acid biosynthesis. The correlation of these events with known hormonal changes is discussed.     

The influence of diet and gastrointestinal fermentation on key enzymes of substrate utilization in marine teleost fishes

Three closely related marine teleosts with similar size, swimming mode, and habitat preference were compared to test the hypothesis that energy metabolism is linked to diet choice in the wild. Key substrate-utilization enzyme activities were assayed from white locomotory muscle and liver in a carnivore (Scorpis violaceus), an omnivore (Girella tricuspidata), and a herbivore (Kyphosus sydneyanus) collected from their natural reef habitat in northeastern New Zealand. The similar energy requirements of the study fishes were reflected in specific enzyme activities of white muscle and suggested high dependence on endogenous fuel that is independent of dietary carbohydrate intake. Clear differences were found in enzymes of hepatic carbohydrate, fat, and ketone body metabolism that appear linked to diet choice and levels of gastrointestinal fermentation. Hepatic metabolism of fat and ketone bodies was also examined in New Zealand samples of the omnivorous Girella cyanea and the herbivorous Kyphosus bigibbus, and the tropical herbivorous species Kyphosus vaigiensis and Kyphosus cinerascens collected from the Great Barrier Reef, Australia. Overall, the results suggest that, like ruminants, herbivorous fishes such as Kyphosus species that rely upon gastrointestinal fermentation preferentially use lipids as major metabolic substrates by an increased capacity for lipid metabolism, and a lower capacity for glycolysis.     

Selective linkage of beta-adrenergic receptors to functional responses in developing rat lung and liver: phosphatidic acid phosphatase, ornithine decarboxylase and lung liquid reabsorption

Neurotransmitter receptors may exhibit transient linkage to specific developmental processes involved in physiological adaptation to extrauterine life and in cell maturation. We have examined the responsiveness of the developing rat lung to beta-adrenergic agonists, using fluid reabsorption, phosphatidic acid phosphatase (an enzyme involved in surfactant synthesis) and ornithine decarboxylase (an enzyme related to cellular development) as markers of these activities. The ability of beta-adrenergic agonists to stimulate phosphatidic acid phosphatase and to cause liquid reabsorption first appeared just before birth, a period in which few receptor binding sites are present; the reactivity of both these processes declined after birth, but the enzymatic stimulation reached a second peak of response during the second and third postnatal weeks. The ability of beta-adrenergic challenge to elicit stimulation of lung phosphatidic acid phosphatase then declined into adulthood, despite the fact that receptor binding sites are increasing during the same period. Lung ornithine decarboxylase activity was poorly linked to beta-receptors in the immediate perinatal period and reached a peak of reactivity during the late postnatal period in which the coupling to phosphatidic acid phosphatase was lost. The pattern for phosphatidic acid phosphatase and liquid content was selective for the lung, as no stimulatory effects were seen for these variables in the liver, despite the comparable beta-adrenergic effects on ornithine decarboxylase in the two tissues. These data suggest that, during development, the coupling of receptors to specific cellular events is more important than the number of receptor sites in determining the pattern of physiological and cellular responses mediated by neurotransmitters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1. The highest blood concentrations of ketone bodies were found at 5 days of age, after which time the concentration fell to reach the adult value by 30 days of age. 2. Both mitochondrial and cytoplasmic hydroxymethylglutaryl-CoA synthase activities were detected, with highest activities being found in the mitochondria at all stages of development. Activity of the mitochondrial enzyme increases rapidly immediately after birth, showing a maximum at 15 days of age, thereafter falling to adult values. The cytoplasmic enzyme, on the other hand, increased steadily in activity after birth to reach a maximum at 40 days of age, after which time activity fell to adult values. 3. Both mitochondrial and cytoplasmic aceto-acetyl-CoA thiolase activities were detected, with the mitochondrial enzyme having considerably higher activities at all stages of development. The developmental patterns for both enzymes were very similar to those for the corresponding hydroxymethylglutaryl-CoA synthases. 4. The activity of heart acetoacetyl-CoA transferase remains constant from late foetal life until the end of the suckling period, after which time there is a gradual threefold increase in activity to reach the adult values. The activity of brain 3-oxo acid CoA-transferase increases steadily after birth, reaching a maximum at 30 days of age, thereafter decreasing to adult values, which are similar to foetal activities. Although at all stages of development the specific activity of the heart enzyme is higher than that of brain, the total enzymic capacity of the brain is higher than that of the heart during the suckling period.     

Variations in ribulose 1,5-bisphosphate carboxylase protein levels,activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.Abbreviations RuBP D-ribulose 1,5-bisphosphate - LTIB low Tris isolation buffer - HTIB high Tris isolation buffer - RIE rocket immunoelectrophoresis     

Enzyme regulation during the synchronous nuclear cycle of Physarum polycephalum: Effects of methotrexate and hydroxyurea on thymidine kinase and deoxycytidine kinase

Specific enzyme activities of thymidine kinase (TK) and deoxy-cytidine kinase (dCK) increase sharply at the onset of synchronous mitosis in macroplasmodia of Physarum polycephalum. They reach a maximum in early S-phase (Physarum lacks a G1 period) and decline to a minimum in late G2. Partial inhibition of DNA synthesis with methotrexate (MTX) or hydroxyurea (HU) retards the onset of the next mitosis and provokes a superinduction of both enzymes, with dCK responding stronger than TK. The temporal pattern observed suggests that the drugs interfere with the postmitotic down-regulation of enzyme expression, possibly due to alterations of the chromatin structure. Moderate inhibition of DNA synthesis still permits the appearance of (delayed) mitoses associated with peaks of enzyme activity at elevated levels. On the other hand, stronger inhibition completely suppresses the onset of mitosis and keeps the enzyme activities at an elevated level without further oscillations. The timing mechanism of periodic enzyme induction therefore appears to be functionally linked to the mitotic signal and does not persist under a stringent DNA block.     

Short-term effects of experimental warming and enhanced ultraviolet-B radiation on photosynthesis and antioxidant defense of <Emphasis Type="Italic">Picea asperata</Emphasis> seedlings

It is generally accepted that sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), granule-bound starch synthases (GBSS) and starch branching enzyme (SBE) play a key role in starch synthesis in wheat grains. Starch synthesis in wheat grains is influenced by genotype and environment. However, what is not known is the degree of variation in enzyme activity during starch accumulation of wheat cultivars differing in kernel types. The present study was carried out to characterize the changing activities of key enzymes during grain filling in two kernel type winter wheat cultivars. Results showed that starch accumulation rate (SAR) and activities of SuSy, AGPase, SSS, GBSS and SBE in large kernel types were significantly higher than those in small kernel types. The soil water deficit experienced during the course of the experiment led to an increase at early grain-filling period and decrease during late grain-filling, respectively, in SAR and activities of key enzymes involved in starch synthesis, especially SuSy, AGPase, SSS, and SBE. Water deficit enhanced grain starch accumulation in small kernel types. It suggests that rainfed treatment increase physiological activities during early grain-filling and promote starch accumulation in small kernel types. The simulation with Richards’ equation showed that it was accumulation duration and SAR that determined the starch accumulation in large kernel types. Compared with small kernel types, plants of large kernel types maintained longer filling duration, higher SAR and greater activities of related enzymes during mid and late grain-filling. These observations suggest stronger sink activities in large kernel types at a later stage of development. Consequently, large kernel types have advantages over the small kernel types in terms of the amount of starch accumulation at mid and late stage, but are sensitive to water deficit.     

Enzyme clusters during the metamorphic period of Ambystoma mexicanum: role of thyroid hormone

Enzyme activities and DNA content have been measure in axolotl liver during the metamorphic period (4-8 months after spawning). Three different types of enzyme activity profiles were observed. In the type I profile (carbamoyl-phosphate synthase, arginase, ornithine transcarbamoylase, and glutamate dehydrogenase) enzyme activity is high in the youngest animals studied, and shows a minimum at 5 months followed by a maximum at 8 months of age. Thereafter activities do not change or slightly decrease. In the type II profile (tyrosine aminotransferase, glucose-6-phosphatase) enzyme activity shows a peak at 5 months of age and is low thereafter. Hexokinase, the enzyme with a type III profile, shows high activity throughout the metamorphic period. DNA content remains high throughout the metamorphic period but decreases 50% between 9 and 12 months of age, probably due to an increase in the size of the hepatocytes. No glucokinase activity was detected. High activities of cluster II enzymes represent early metamorphic events, while the rising part of cluster I is associated with late metamorphic events. The apparent molecular specific activity increases during natural development between 5 and 9 months of age, or precociously, upon thyroid hormone treatment. This change in apparent molecular specific activity is correlated to the advent of ureotelism.     

Differential action of thyroid hormones on the activity of certain enzymes in rat kidney and brain.

In rat kidney several mitochondrial and soluble enzyme activities are stimulated by thyroid hormones and the mitochondrial membrane fluidity is also increased. However, the ketone metabolism enzyme activities of D-3-hydroxybutyrate dehydrogenase and of 3-oxoacid CoA-transferase are not significantly affected by the hyperthyroid state and the ketone body concentration is not greatly changed. Therefore, in hyperthyroid rats the response of the kidney, as far as the ketone bodies and their metabolizing enzymes are concerned, is at variance with that of the liver and the heart. In the brain of young rats, age 8-9 weeks, the activities of the enzymes of ketone body metabolism and those responsible for other metabolic pathways are not influenced by the hyperthyroid state. In these animals, however, the activities of two enzymes, NAD-isocitrate dehydrogenase and pyruvate kinase, are still stimulated by 28 and 41%, respectively. This can be probably related to the higher energy requirement for definitive brain maturation in young hyperthyroid rats.     

早晚季不同耐光氧化特性水稻品种籽粒灌浆关键酶活性变化

选用光氧化敏感和不敏感的两大类型4个水稻品种研究早晚季耐光氧化反应特性与其稻米品质稳定性密切相关的生理生化原因.结果表明,耐光氧化反应特性强的适强光生态型水稻品种的平均灌浆速率(G_mean)、达最大灌浆速率时间(T_max.G)和达最大灌浆速率百粒重(W_max.G)等参数在早晚季灌浆结实期间保持相对稳定;而对光氧化敏感的弱光生态型品种佳禾早占以上参数的早晚季稳定性较差.比较早晚季籽粒灌浆充实过程中关键酶活性的变化结果可见,耐光氧化反应特性强的品种,灌浆速率高峰到灌浆终期ADPG-焦磷酸化酶和淀粉合成酶活性早晚季变异比耐光氧化反应特性弱的品种小,且耐光氧化反应特性强的品种灌浆高峰前后酶活性变异的幅度早晚季也较一致.     

Prosopis juliflora pollen allergen induced hypersensitivity and anaphylaxis studies in guinea pigs

In vivo and in vitro allergenic activities of Prosopis juliflora pollen allergens were measured in guinea pigs. Intracutaneous skin test showed an early wheal flare response and a late erythema-redness, sensitized with various concentrations (100, 50, 25, 5 and 1.5 micrograms/ml) of Prosopis juliflora pollen extract after administration of a challenging dose. A 50 micrograms/ml sensitizing dose of Prosopis juliflora pollen allergen gave optimum skin response as both early and late effects. The nature of immunochemical reactivity between pollen allergens and reaginic antibodies were further characterized by histamine release test, gel diffusion test, radioallergosorbent test and passive cutaneous anaphylaxis test. These tests confirm allergenicity caused by Prosopis juliflora pollen allergens and showed the binding of allergens with reaginic antibody and its regulation in guinea pigs.