Adaptive Enzyme Research Articles

Glutathione dependent enzymes and antioxidant defences in truffles: organisms living in microaerobic environments

Truffles live in an environment poor in oxygen, and are a good model for investigation of enzymatic adaptation to microaerobic conditions. We studied the antioxidant and glutathione dependent enzymatic endowment of Tuber truffles. Superoxide dismutase, catalase, glutathione peroxidase Se dependent, glutathione reductase, glyoxalase 1 and glyoxalase 2 are expressed and correlate with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles. A very low or undetectable glutathione S-transferase activity was found. For comparison the same enzyme activities were investigated in Agaricus bisporus which is epigeous and non-mycorrhizal.

Extracellular hydrolytic enzymes in the fungal genus Verticillium: adaptations for pathogenesis

The insect and plant pathogens within the fungal genus Verticillium showed enzymatic adaptation (production and regulation) directed to the degradation of some of the polymers found in the integument of their respective hosts. For example, the facultative plant pathogens (V. albo-atrum and V. dahliae) produced greater levels of cellulase and xylanase than the facultative insect pathogen (V. lecanii). Verticillium lecanii produced extracellular subtilisin-like protease when grown in insect cuticle medium but not in plant cell wall medium, while the plant pathogen V. albo-atrum showed a diminished regulatory component in the production of this enzyme. The opportunistic pathogens (V. fungicola and V. coccosporum) and the saprobic species (V. rexianum) were less specific in the production and regulation of several proteases as well as cellulases and xylanases. A dendrogram based on cluster analysis compiled from fungal API-ZYM profiles showed commonalties in a broad array of extracellular enzymes within a host-pathogen group (i.e. insect or plant pathogen). The opportunistic pathogens were dispersed throughout the dendrogram, suggestive of the diversity in type and expression of extracellular enzymes.Key words: extracellular enzymes, pathogenic fungi.

Extracellular hydrolytic enzymes in the fungal genus <i>Verticillium</i>: adaptations for pathogenesis

The insect and plant pathogens within the fungal genus Verticillium showed enzymatic adaptation (production and regulation) directed to the degradation of some of the polymers found in the integume...

Human skeletal muscle pyruvate dehydrogenase kinase activity increases after a low-carbohydrate diet.

To characterize human skeletal muscle enzymatic adaptation to a low-carbohydrate, high-fat, and high-protein diet (LCD), subjects consumed a eucaloric diet consisting of 5% of the total energy intake from carbohydrate, 63% from fat, and 33% from protein for 6 days compared with their normal diet (52% carbohydrate, 33% fat, and 14% protein). Biopsies were taken from the vastus lateralis before and after 3 and 6 days on a LCD. Intact mitochondria were extracted from fresh muscle and analyzed for pyruvate dehydrogenase (PDH) kinase, total PDH, and carnitine palmitoyltransferase I activities and mitochondrial ATP production rate (using carbohydrate and fat substrates). beta-Hydroxyacyl CoA dehydrogenase, active PDH (PDHa), and citrate synthase activities were also measured on whole muscle homogenates. PDH kinase (PDHK) was calculated as the absolute value of the apparent first-order rate constant of the inactivation of PDH in the presence of 0.3 mM Mg2+-ATP. PDHK increased dramatically from 0.10 +/- 0.02 min-1 to 0.35 +/- 0.09 min-1 at 3 days and 0.49 +/- 0. 06 min-1 after 6 days. Resting PDHa activity decreased from 0.63 +/- 0.17 to 0.17 +/- 0.04 mmol. min-1. kg-1 after 6 days on the diet, whereas total PDH activity did not change. Activities for all other enzymes were unaltered by the LCD. In summary, severe deficiency of dietary carbohydrate combined with a twofold increase in dietary fat and protein caused a rapid three- to fivefold increase in PDHK activity in human skeletal muscle. The increased PDHK activity downregulated the amount of PDH in its active form at rest and decreased carbohydrate metabolism. However, an increase in the activities of enzymes involved in fatty acid oxidation did not occur.

Mitochondrial oxygen consumption, lipid peroxidation and antioxidant enzyme systems in skeletal muscle of senile dystrophic mice.

To evaluate age-dependent abnormalities in mitochondrial redox metabolism specifically in dystrophic skeletal muscle, oxygen consumption, thiobarbituric acid reactivity (TBARS), free-radical scavengers and oxidative marker enzymes were measured in the skeletal muscle from adult and senile control (C57BL/10) and dystrophic (mdx/+) mice. Mitochondrial oxygen consumption in state 3 was significantly lowered with age in the senile dystrophic (-52%) and less markedly in the senile control (-30%) skeletal muscle. Compared with adult muscle, mitochondrial concentration of TBARS and cellular concentration of lipofuscin were significantly increased in senile control and dystrophic muscle. Enzymatic activity of glutathione peroxidase (GSH-Px) and concentration of alpha-tocopherol were significantly increased in the senile control (GSH-Px 43+/-5.7 vs 53+/-8.7 U/g protein, alpha-tocopherol 0.19+/-0. 09 vs 0.29+/-0.14 micromol/g total lipids), but significantly decreased in the senile dystrophic (GSH-Px 80+/-8.0 vs 53+/-12 U/g protein, alpha-tocopherol 0.45+/-0.13 vs 0.19+/-0.03 micromol/g total lipids) muscle. Selenium content was significantly decreased only in senile dystrophic muscle (1.37+/-0.42 vs 0.78+/-0.21 nmol/g wet muscle). In conclusion, the enzymatic adaptation to reactive oxygen species was limited in the dystrophic skeletal muscle, suggesting a higher need for antioxidants, especially alpha-tocopherol.

Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes.

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (-1.86 to 1 degree C) and three South American (4 to 10 degree C) notothenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs.

The Role of Adaptive Enzymes in Carbohydrate Oxidation by Stressed and Senescing Plant Tissues

The Role of Adaptive Enzymes in Carbohydrate Oxidation by Stressed and Senescing Plant Tissues

THE TWO-COMPONENT SIGNALING PATHWAY OF BACTERIAL CHEMOTAXIS: A Molecular View of Signal Transduction by Receptors, Kinases, and Adaptation Enzymes

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

Muscle enzyme activity in humans: role of substrate availability and training.

To study the effect of nutrient intake (substrate flux) and training on muscle enzyme activities, 36 untrained healthy men adapted for 7 wk to a fat-rich or a carbohydrate-rich diet. Ten of the 18 subjects on each diet completed an endurance training program, and the remaining 8 served as controls. Maximal oxygen uptake was increased (11%) in the trained groups (P < 0.05). Irrespective of training, beta-hydroxyacyl-CoA-dehydrogenase activity in the vastus lateralis muscle was significantly increased by an average of 25% after adaptation to a fat-rich diet and was unchanged after adaptation to a carbohydrate-rich diet. In contrast, irrespective of diet, muscle citrate synthase activity and hexokinase activity were increased (P < 0.05) after adaptation to training by 17 and 18% in the group fed the carbohydrate, rich diet and by 17 and 12% in the group fed the fat-rich diet, respectively, and were unchanged in the two control groups. We suggest that diet can affect muscle enzymatic adaptation, presumably through an effect on the substrate flux.

Catalytic Properties of Two Pyruvate Kinase Isoforms in Nordic Krill, Meganyctiphanes norvegica, With Respect to Seasonal Temperature Adaptation

Two isoforms of pyruvate kinase (PK I and PK II) were partly purified and characterized from the Nordic krill Meganyctiphanes norvegica. Both PK variants were present in summer and winter specimens with a tissue specificity in abdominal muscle (PK I) and cephalothorax (PK II). Obvious differences were found in chromatographic and kinetic characteristics. Enzymatic adaptations to low temperatures were found in PK I only, whereas PK II did not contribute to seasonal temperature adaptation. In winter specimens, the activation energy of PK I decreased significantly from 53.2 ± 1.5 to 50.2 ± 1.2 kJ·mol−1. The affinity of PK I to phosphoenol-pyruvate was higher in winter (KM = 0.024 ± 0.002 mmol·l−1) compared to summer (KM = 0.033 ± 0.003 mmol·l−1). Both effects lead to an increased efficiency of this enzyme isoform in the cold. In contrast, KM values of PK II showed no significant differences between summer (KM = 0.181 ± 0.014 mmol·l−1) and winter specimens (KM = 0.193 ± 0.015 mmol·l−1). The effects of cooperativity remained unchanged during the seasons with approximate values of nHill = 1.0 (PK I) and 1.5 (PK II). Fructose-1,6-bis-phosphate affected only PK II by shifting sigmoidal kinetics to hyperbolic curves resulting in a decrease of KM to 0.027 mmol·l−1. Further effectors were tested showing an inhibiting effect of oxalate on both isoforms with a reduction to 20% and 50% in PK I and PK II, respectively. Presumably, the ecophysiological effect of the capacity to regulate muscle PK is related to the necessity to increase motility during vertical migration and phases of feeding activity.

Chronic enhancement of neuromuscular activity increases acetylcholinesterase gene expression in skeletal muscle.

We determined levels of mRNA encoding acetylcholinesterase (AChE) in muscles of rats subjected to chronic enhancement of neuromuscular activation. After 8 wk of voluntary wheel running, extensor digitorum longus (EDL) muscles displayed a 72% increase in total AChE activity as a result of a selective threefold increase in the G4 content. Soleus muscles, on the other hand, exhibited a 30% decrease in A12 while displaying a small (33%) increase in total AChE activity. These enzymatic adaptations were paralleled by increases in the levels of AChE mRNAs in both EDL (32%; P < 0.03) and soleus (42%; P < 0.02) muscles. In addition, compensatory hypertrophy of the plantaris muscle increased total AChE activity by 75%. This change was reflected by an elevation in all AChE molecular forms with A12 (89%) and A8 (179%) showing the most prominent increases. Similar to exercise-trained muscles, hypertrophied plantaris muscles displayed an increase in AChE transcripts (25%; P < 0.04). These results indicate that increases in neuromuscular activity modulate expression of the AChE gene in vivo and suggest the involvement of pretranslational regulatory mechanisms in the adaptive response of AChE to enhanced neuromuscular activation.

Physiological Adaptations in the Gastrointestinal Tract of Crayfish

SYNOPSIS. Crayfish are the dominant macrocrustacean in many aquatic ecosystems and are the largest crustacean aquacultural industry in the United States, yet we know relatively little about their preferred and nutritionally important foods, as well as their ability to utilize those foods. This review focuses on the ability of crayfish to detect foods, reduce food particle size, digest macronutrients and the control of those functions. Of particular interest are the enzymatic capabilities of crayfish, especially trypsin, an alkaline protease, cellulase, muramidase, and possibly chitinase and chitobiase. The coordinated neural control of crayfish food location, ingestion and movement has been well documented, while hormonal control mechanisms have not. The conclusion we must draw from our current state of knowledge is that crayfish have ample abilities to taste and locate potential foods and enzymatic adaptations developed in crayfish that allow use of many of the foods they encounter in a benthic aquatic environment; other adaptations are lacking or have not been elucidated.

Oxidative Stress: Pathogenetic Mechanisms

Radical and nonradical ‘reactive oxygen species (ROS)’ are normally occurring intermediates of aerobic cellular metabolism. Under physiologic conditions, they are kept from causing noticeable damage by antioxidant enzymes and small molecular weight compounds. Oxidative stress develops when antioxidants are overwhelmed by ROS. This may occur via overproduction of the latter and/or by deficiency of antioxidant defense. ROS in excess, especially the highly reactive hydroxyl radical (HO·), which is usually generated with the catalytic help of transition metals such as ferrous ions, can attack virtually all biological structures. Consequences may be functionally relevant when sensitive macromolecules are altered by ROS, in particular DNA (e.g. mutations, base modifications), proteins (formation of disulfide bridges etc.), lipids (lipid peroxidation), and polysaccharides (depolymerisation). Type and severity of the damage and cellular responses to it, including adaptive enzyme induction, determine the outcome in terms of cellular dysfunction or death. Many human disorders are thought to be associated with oxidative stress. ROS seem to play a predominant role, for instance, in radiation effects, damage by oxidant chemicals (many poisons and chemotherapeutic drugs), lung lesions due to hyperbaric oxygen, and chronic deficiencies of selenium and vitamin E. Oxidative stress is also thought to be important in other diseases and processes, e.g. in ischemia-reperfusion damage, central nervous system injury and stroke, severe chronic inflammatory disorders (for example rheumatoid arthritis), the development of arterial atherosclerosis, cancero-genesis, and ageing.

Studies on beta-galactosidase and their impact on molecular biology: a personal view.

Studies on beta-galactosidase and their impact on molecular biology: a personal view.

Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae

Variations in some enzyme activities during larval development of sea bass fed live prey were investigated from hatching to day 40. Fluctuations in the enzyme specific activities (except for trypsin) occurred in three phases: initially a sharp increase until day 12, followed by a plateau and subsequently a decrease around day 23. Then activities remained constant until day 40. Trypsin activity kept rising until day 23, then fell. Enzymatic adaptation to a change in diet was studied by feeding larvae with microparticulate diet from day 25. Adaptation to dietary change was observed for amylase, alkaline phosphatase and leucine aminopeptidase, assayed in whole larvae. In larvae fed microparticulate dry diet, the activities of these three enzymes tended to be higher than in those fed natural prey. Although poor growth was observed in larvae fed microparticles, the brush border enzyme activities purified from whole body homogenate, were not impaired.

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.

Glucose transporters and maximal transport are increased in endurance-trained rat soleus.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantum molecular computing: The self-assembly model

The principle of macromolecular self-assembly is used to construct a model of computing that exploits quantum effects to achieve enhanced real-time capabilities. Signals impinging on a device (or biological cell) trigger the appearance of macromolecules that self-assemble into a mosaic. Adaptor enzymes recognize features of the mosaic and link these to the output of the device. In this way, a symbolic pattern recognition problem is converted to a free-energy minimization process. A Hartree-type self-consistent-field formalism is developed for treating the self-assembly process. The formalism demonstrates that the parallelism inherent in the quantum mechanical wave function (the superposition of electronic states) can speed up the exploration of the potential surface, thereby increasing computational search power over what can be achieved with conventional models of computation. © 1992 John Wiley & Sons, Inc.

Myocardial Lactate Dehydrogenase and its Isoenzyme Activities in Transplanted Human Hearts

Lactate dehydrogenase and its heart (H) and muscle (M) subunit activities were studied in right ventricular endomyocardial biopsies from eight transplanted human hearts and compared with five chronically failing hearts and six normal human hearts from brain-dead liver/kidney donors. Of the 17 transplant biopsies (taken 5-95 weeks postoperatively), only two showed histologic signs of chronic rejection: They were excluded from the group analysis. A higher proportion of the M subunit of lactate dehydrogenase (M%) was found in the transplanted and the chronically failing hearts than in the normal hearts, presumably reflecting increased myocardial anaerobic glycolytic stress. In the early post-transplantation period, M% was higher in the transplanted than in the chronically failing hearts. Thereafter M% gradually fell, but had not reached normal levels 1-2 years after transplantation. During that time it was similar to the values in the chronic-failure hearts. In the two biopsies with chronic rejection, M% was nearly twice as high as in contemporaneous biopsies showing mild or no rejection. Monitoring of enzymatic adaptation from endomyocardial biopsies may be of clinical interest.

Enzymatic adaptations to treadmill training under the influence of naftidrofuryl acid in diaphragm and limb muscles of old rats

The effects of training and naftidrofuryl treatment were observed in 21-month-old Long-Evans rats. Rats were injected intraperitoneally twice daily for 8 weeks with 7 mg.kg-1 of naftidrofuryl acid (SN, TN), or with 7 mg.kg-1 fumaric acid (SC and TC) or used as solvent. Training groups (TC, TN) started a progressive 8-week training programme of treadmill exercise. The activities of lactate dehydrogenase (LDH), hexokinase (HK), citrate synthase (CS) and 3-hydroxyacyl-Co-A-dehydrogenase (HAD), were measured in Soleus (SOL), Extensor Digitorum Longus (EDL) and Diaphragm (DIA) muscles. The mean VO2max value was 65 ml.min-1.kg-1 for 21-month-old rats. The training protocol induced increases in the mean VO2max values in the TC and TN groups, 71.8 and 74.4 ml.min-1.kg-1. In sedentary groups (SN), naftidrofuryl increased enzymatic activities (HK, CS, HAD) in the three muscles examined. When the animals underwent 8 weeks of physical training, the enzymatic activities (HK, CS, HAD) increased in SOL, EDL and DIA. When training was combined with naftidrofuryl treatment the increases in enzymatic activities were greater than those induced by training alone. However, the total changes did not differ for the sum of the changes produced by each condition alone.