Temperature and the catalytic activity of enzymes: A fresh understanding

The discovery of an additional step in the progression of an enzyme from the active to inactive state under the influence of temperature has led to a better match with experimental data for all enzymes that follow Michaelis–Menten kinetics, and to an increased understanding of the process. The new model of the process, the Equilibrium Model, describes an additional mechanism by which temperature affects the activity of enzymes, with implications for ecological, metabolic, structural, and applied studies of enzymes.     

Effect of enzyme concentration on apparent specific activity of hydrogenase

The effect of enzyme concentration on the H2-uptake and H2-evolving activities of the reversible hydrogenase from Thiocapsa roseopersicina was examined. In the activity range assayed by a spectrophotometric technique the apparent H2-uptake specific activity varied greatly with hydrogenase concentration. Study of H2-evolving activity measured by the H2 electrode method and compared with a gas chromatographic assay also indicated that specific activity was highly dependent on enzyme concentration. The results indicate that the widely applied hydrogenase assays give systematically erroneous specific activity values. These assays should be used only for relative measurements and the hydrogenase concentration in the reaction mixture should be kept constant. To make the data from various laboratories comparable the assay parameters should be standardized.     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

Model of a hypothetical protohominid dentition and its implications for hominid phylogeny

The complete dentition of the common ancestor ofAustralopithecus andHomo, intermediate between that of a pongid and a hominid, is virtually unknown. The maxillary dentition (P3-M2) ofRamapithecus brevirostris Lewis, 1934, a pongid from the Early Pliocene, and that of hominids from the Late Pliocene and Plio/Pleistocene is known. SinceR. brevirostris is probably ancestral to the hominids, a model of intermediate maxillary dentition (P3-M2) is extrapolated and described. The model represents a hypothetical protohominid dentition. It does not conform with the teeth ofAustralopithecus, but shows greater morphological affinity to hominine dentition and to 5 myo hominids. TheHomo lineage, therefore, may go back to the Middle Pliocene. According to the normal sequence of evolution, it is most unlikely thatAustralopithecus gave rise toHomo, but much more probable that a very early, generalizedHomo evolved into an advanced, specializedAustralopithecus.     

The effect of temperature on glycollate decarboxylation in leaf peroxisomes

[1-¹⁴C]glycollate was oxidised to¹⁴CO₂ by peroxisomes isolated from leaves of spinach beet about 3 times as rapidly at 35°C as at 25°C; the rate was further increased with rise in temperature to a maximum at 55°C. These increases are shown to be mainly due to the increased H₂O₂ available to oxidise glyoxylate non-enzymically as a result of the higher temperature coefficient of glycollate oxidase activity relative to that of catalase. These results are compared with similar increases in the rate of¹⁴CO₂ release between 25°C and 35°C when [1-¹⁴C]glycollate was supplied to leaf discs in light or darkness. The role of these reactions in accounting for the temperature effect on the release of photorespiratory CO₂ is discussed.Abbreviations PHMS Pyrid-2-yl--hydroxymethane sulphonate - FMN flavin mononucleotide     

The effect of urea exposure on isoaspartyl content and protein l-isoaspartate methyltransferase activity in Drosophila melanogaster

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (β-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein l-isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.     

Expression of GUS in primary transformants and segregation patterns of GUS,TL- and TR-DNA in the T1 generation of hairy root transformants ofLotus corniculatus

Agrobacterium rhizogenes was assessed as a vehicle for transformation ofLotus corniculatus. Plants were co-transformed usingA. rhizogenes strain LBA 9402 harbouring the bacterial plasmid pRi1855 and the binary transformation vector pJit 73. pRi 1855 transfers both T_L and T_R sequences, while pJit 73 encodes β-glucuronidase (GUS) and also two selectable marker genes giving resistance to the antibiotics kanamycin and hygromycin. Three primary transformants (lines 1,6 and 12) were subjected to detailed morphological and biochemical analysis and lines 6 and 12 were also analysed at the molecular level. Tissues of both lines 6 and 12 were resistant to hygromycin and expressed GUS. Analysis of various tissues of each line showed a significantly lower GUS activity in line 6 than in line 12. Genetical analysis of progeny produced between control plants and lines 6 and 12 indicated that line 6 had one dose of theuid gene while line 12 had two or more independently segregating doses of the gene. Both lines 6 and 12 contained multiple copies of T_L-DNA, while only line 6 was T_R positive. In the progeny of lines 6 and 12 there was no evidence for linkage of T_L-DNA withuid, while in the progeny of line 6, T_R-DNA was under-represented. GUS-positive progeny which were free of both T_L and T_R sequences were identified from both lines.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

The effect of temperature on the development time and brood size of diaptomus pallidus herrick

Diaptomus pallidus individuals were raised in the laboratory at three temperatures (15, 20, and 25°C) and fed an alfalfa and trout-food diet ad libitum. Data were taken on the development times of the egg, naupliar, and each copepodid stage and the brood sizes of field animals acclimated to the test conditions.The results indicated D. pallidus does not have a temperature range over which its development rate is nearly constant as earlier reported. Rather, the development rate is temperature dependent within the experimental range. Broods produced at 20°C and 25°C were significantly smaller than those produced at 15°C but not significantly different from each other.     

The effect of daily exposure to low hardening temperature on plant vital activity

Phenomenological responses of plants to daily short-term exposure to low hardening temperature was studied under chamber and field conditions. Experiments were carried out on cucumber (Cucumis sativus L.), barley (Hordeum vulgare L.), marigolds (Tagetes L.), and petunia (Petunia × hybrida) plants. The obtained data demonstrated a similar pattern of response in all studied plant species to different variants of exposure to low hardening temperature. The main features of plant response to daily short-term exposure to low hardening temperature include: a higher increment in cold tolerance (cf. two-or threefold increase relative to constant low hardening temperature) that peaked on day 5 (cf. day 2 at constant low hardening temperature) and was maintained for 2 weeks (cf. 3–4 days at constant low hardening temperature); a simultaneous increase in heat tolerance (cf. twofold relative to constant low hardening temperature) maintained over a long period (cf. only in the beginning of the exposure to constant low hardening temperature); a sharp drop in the subsequent cold tolerance after plant incubation in the dark (cf. a very low decrease in cold tolerance following the exposure to constant low hardening temperature); a combination of high cold tolerance and high photochemical activity of the photosynthetic apparatus (cf. a low non-photochemical quenching at constant low hardening temperature); and the capacity to increase cold tolerance in response to repeated short-term exposures to low hardening temperature in plants grown outdoors (cf. a gradual increase after repeated exposure to constant low hardening temperature). Possible mechanisms underlying the plant response to daily short-term exposure to low temperature are proposed.     

Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in twoFusarium species

Among all antibiotics tested, amoxycillin (500 ppm) completely inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activity inF. oxysporum; none of the antibiotics did so inF. moniliforme. No antibiotic completely inhibited the cellulase activity in both test organisms, however, amoxycillin was better than other antibiotics in inhibiting the cellulase activity in both the organisms.     

Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases

During the biosynthesis of heme d₁, the essential cofactor of cytochrome cd₁ nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.     

Cryopreservative effect of leupeptin on early human bone marrow progenitors

Summary Leupeptin, a thiol- and serine-proteinase inhibitor of low molecular weight, quickly enters viable cells. This property has been used to protect cells during thawing against intracellular proteolytic activities released by injured lysosomes. The bone marrow nucleated cells were frozen without rate-controlled freezing devices. Concentrations ranging from 0.1 to 1μM of leupeptin allow to recover 87% of the most immature multipotent bone marrow progenitors which can develop in vitro into large multilineage colonies, instead of 58% recovery without leupeptin. The protective effect of leupeptin is particularly useful to freeze cells difficult to cryopreserve or when freezing-control equipments are not available.     

Mechanisms regulating GABAergic inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety disorders

Summary. The amygdala, a temporal lobe structure that is part of the limbic system, has long been recognized for its central role in emotions and emotional behavior. Pathophysiological alterations in neuronal excitability in the amygdala are characteristic features of certain psychiatric illnesses, such as anxiety disorders and depressive disorders. Furthermore, neuronal excitability in the amygdala, and, in particular, excitability of the basolateral nucleus of the amygdala (BLA) plays a pivotal role in the pathogenesis and symptomatology of temporal lobe epilepsy. Here, we describe two recently discovered mechanisms regulating neuronal excitability in the BLA, by modulating GABAergic inhibitory transmission. One of these mechanisms involves the regulation of GABA release via kainate receptors containing the GluR5 subunit (GluR5KRs). In the rat BLA, GluR5KRs are present on both somatodendritic regions and presynaptic terminals of GABAergic interneurons, and regulate GABA release in an agonist concentration-dependent, bidirectional manner. The relevance of the GluR5KR function to epilepsy is suggested by the findings that GluR5KR agonists can induce epileptic activity, whereas GluR5KR antagonists can prevent it. Further support for an important role of GluR5KRs in epilepsy comes from the findings that antagonism of GluR5KRs is a primary mechanism underlying the antiepileptic properties of the anticonvulsant topiramate. Another mechanism regulating neuronal excitability in the BLA by modulating GABAergic synaptic transmission is the facilitation of GABA release via presynaptic α_1A adrenergic receptors. This mechanism may significantly underlie the antiepileptic properties of norepinephrine. Notably, the α_1A adrenoceptor-mediated facilitation of GABA release is severely impaired by stress. This stress-induced impairment in the noradrenergic facilitation of GABA release in the BLA may underlie the hyperexcitability of the amygdala in certain stress-related affective disorders, and may explain the stress-induced exacerbation of seizure activity in epileptic patients.     

The effect of temperature on maturation threshold body-length in Daphnia magna

Aquatic invertebrates are usually larger at maturity when water temperatures are lower. For the freshwater cladoceran Daphnia, it has been suggested that a threshold size must be attained to initiate maturation, which results two instars later in the deposition of eggs into the brood chamber. This threshold size is believed to temperature on maturation threshold body-length in Daphnia magna. Daphnids were raised from birth to maturity under three constant-temperature regimes (12°C, 16°C, 22°C), and two food-level conditions. Animals were measured daily, and a body-length based maturation threshold determined for each individual. We demonstrate that mean maturation threshold length is negatively correlated with ambient water temperature. Further, daphnids with a larger threshold length tended to be larger at maturity. A maturation threshold linked to body length suggests that reduced variation in size at maturity is adaptive, even at the cost of additional variation in instar number or age at maturity.     

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q₁₀ and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K_m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V_m versus 1/6PG and 1/NADP plots gave a V_m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K_i of 31.91±1.31 μM. The relatively small K_i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.     

The Effects of Triiodothyronine on Rat Testis: A Morphometric and Immunohistochemical Study

The aim of present study was to investigate the effects of 3,3′,5-triiodothyronine (T₃) on rat testis both morphometrically and immunohistochemically with determining of insulin-like growth factor I (IGF-I) expression. Adult male Wistar-albino rats used in the study were divided into two groups; control and T₃-treated groups. After T₃ treatment there was observed to be a decrease in testicular weights, diameters of seminiferous tubules and the number of sertoli cells, and an increase in the number of leydig cells (P<0.05). Some of the seminiferous tubule lumens of T₃ administrated rats had cellular debris. IGF-I was localized in sertoli cells, late spermatids and leydig cells of all groups. IGF-I immunoreactivity in T₃ treated rats was higher than in controls in all stages of the cycle of rat seminiferous epithelium, but the staining intensity of leydig cells were similar in both groups. In conclusion, the present results suggest that T₃ may modulate the testicular function by affecting IGF-I activity at the gonadal level.     

Beijerinckia indica var.penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity

Summary Beijerinckia indica var.penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, -irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.     

The effect of temperature on salt uptake by tomato plants with diurnal and noctural waterlogging of salinized rootzones

Summary Tomato plants were grown at three constant temperatures (10, 20 and 28°C) with drained or waterlogged rootzones and were irrigated with saline solution (0.09M NaCl).Each increase in temperature resulted in an increase in leaf Na-ion and Cl-ion concentrations in plants grown with drained rootzones. However, with plants grown with waterlogged rootzones, maximum leaf concentrations of Na-ions and Cl-ions occurred at 20°C.At 10°C there were no differences between Na-ion and Cl-ion concentrations for drained or waterlogged treatments. At 20 and 28°C, waterlogging of the rootzone resulted in significantly higher concentrations of Na-ions and Cl-ions in leaf and stem tissues than occurred with drained rootzones.There were no differences in Na-ions and Cl-ions and Cl-ions in plant tops if plants were waterlogged with saline solution during the day or night.Transpiration increased significantly with each increase in temperature but showed no other treatment dependent responses.     

Ammonia and light effect on nitrogenase activity in nitrogen-limited continuous cultures of Rhodopseudomonas capsulata. Role of glutamine synthetase

Nitrogen-limited continuous cultures of Rhodopseudomonas capsulata were used to investigate some aspects of the regulation of nitrogenase activity. The role of glutamine synthetase (GS) in this regulation was examined by measuring changes of its adenylylation state when the light intensity and the nitrogen source were varied. Maximal nitrogenase activity was observed at a dilution rate corresponding to about one third of the maximum specific growth rate (_max), both in ammonia- and in glutamate-limited cultures. At higher dilution rates, both GS and nitrogenase were inactivated by ammonia. Determination of the kinetics of inhibition of both enzymes indicated that the degree of inactivation of nitrogenase and the adenylylation state of GS were not closely related. Increase of light intensity stimulated nitrogenase activity dramatically. Conversely, a shift-down in light intensity to a limiting value resulted in a decrease of nitrogenase activity suggesting that synthesis was inhibited. On the other hand, the adenylylation state of glutamine synthetase appeared to be unaffected by changes in light intensity, indicating that GS is probably not involved in the regulation of nitrogenase expression by light.Abbreviations GS glutamine synthetase - R Rhodopseudomonas - Rs. Rhodospirillum - CTAB cetyltrimethylammonium bromide Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday