Effects of beta blockade and atropinisation on plasma catecholamine concentration during exercise.

The changes in plasma catecholamine concentration (deltaC) following beta-blockade (practolol, 15 mg) and atropinisation (Atropine, 1.8 mg) have been studied on 5 healthy male subjects during exercise on a motor driven treadmill. The results showed that for a given V-2 and % VO2 max, beta blockade was without effect on delta C (except in one athletic subject, but atropine produced a rise in deltaC. In relation to Q, both drugs produced an increase in deltaC, but for a given cardiac frequency (fH) deltaC was higher with beta blockade, and lower with atropinisation than found in control experiments. The intra- and inter-subject variability of delta C in relation of fH was resolved by considering the change in cardiac frequency calculated from baseline value obtained during walking at 6.44 km/h on the level, and expressed as a percentage of the maximal fH attainable for given individuals under the different drug and control conditions (%deltafH). It was concluded that during short term exercise, the rise of deltaC in relation to %deltafH reflects both the myocardial sensitivity to vagal and beta blockade, and the circulatory vasoconstrictor control of blood vessels which is required to sustain increases in systemic and muscle blood flow.     

Neonatally-induced thermotolerance: physiological responses

1. Broiler cockerels exposed to neonatal heat (35-37.8 degrees C for 24 hr) at 5 days of age experienced significantly lower mortality upon exposure to elevated temperatures (35-37.8 degrees C) at 43 days of age than did cockerels not given neonatal heat exposure. 2. No differences were found between neonatally stressed and neonatal control groups in water consumption, core and surface temperature, plasma T3 and T4, protein or glucose concentration when exposed to juvenile heat stress. 3. Heterophil to lymphocyte ratios were significantly lower in the neonatally stressed than in the neonatal control group when exposed to heat at 44 days of age. 4. The results indicate that lower mortality during periods of high environmental temperature in the neonatally stressed chicks may involve homeostatic mechanisms different from those utilized during acclimation to high environmental temperature.     

Solubilization of the chemokine receptor CXCR4

The chemokine receptor CXCR4 was solubilized from the human T-cell line CEM by using the detergent n-dodecyl-beta-maltoside (DDM) and cholesteryl hemisuccinate ester (CHS). Binding studies with (125)I-SDF-1alpha revealed a dissociation constant of 5.33 nM and a receptor density (B(max)) of 2.68 pmol/mg in CEM membranes at 4 degrees C. The affinity of solubilized CXCR4 for SDF-1alpha was identical to membrane-bound CXCR4. Binding of gp120 to solubilized CXCR4 was demonstrated by coprecipitation of gp120 with anti-CXCR4 antibodies.     

Peripheral-type benzodiazepine receptors in human blood cells of patients affected by migraine without aura

The kinetic parameters at equilibrium of peripheral benzodiazepine receptors in platelets, lymphocytes and granulocytes of 15 patients affected by migraine without aura were tested using [3H]PK 11195, a specific radioligand for this receptor and compared with the same number of healthy controls: a statistically significant increase (platelets 212%, lymphocytes 203%, granulocytes 171%, as absolute percentage) in the maximal number of binding sites (B(max)) in all three patient samples, compared with healthy controls was detected; on the contrary, the values of the dissociation constant (K(d)) at equilibrium do not show any statistically significant variations between the two groups. These data further confirm the presence of peripheral biochemical alterations in migraine without aura. As peripheral benzodiazepine receptors appear to be involved in the regulation of the mitochondrial respiratory chain, the observed increase in B(max) might be related to the mitochondrial anomalies found in migraine disorders.     

The effect of temperature acclimation on myocardial beta-adrenoceptor density and ligand binding affinity in African catfish (Claris gariepinus)

This study assessed the effects of temperature acclimation on myocardial beta-adrenoceptor density (B(max)) and binding affinity (K(d)) in African catfish (Claris gariepinus) acclimated to 15, 22 and 32 degrees C. B(max) values were not significantly different (P > 0.05) among the three acclimation groups. Conversely, the K(d) value of the 32 degrees C acclimation group (K(d) = 0.88) was significantly higher (P = 0.002) than both the 15 degrees C (K(d) = 0.48) and 22 degrees C (K(d) = 0.46) acclimation groups. In addition, K(d) of rainbow trout (Oncorhynchus mykiss) was significantly lower (P < 0.001) and B(max) significantly higher (P < 0.05) than that of African catfish at all three acclimation temperatures. These results contrast with those reported previously for temperate species, in which B(max) is inversely related to acclimation temperature, and counter a previous suggestion that B(max) is higher in tropical versus temperate species.     

γ-Aminobutyric Acid: Temperature Dependence in Benzodiazepine Receptor Binding

The effects of muscimol and/or incubation temperature on the inhibition of [3H]flunitrazepam receptor binding by benzodiazepine receptor ligands were investigated. At 0 degree C muscimol decreased the Ki values for some ligands as displacers of [3H]flunitrazepam binding to brain-specific sites while increasing or having no effect on the Ki values for other ligands. The Ki values for some ligands are higher at 37 degrees C than at 0 degree C but are reduced by muscimol at both 0 degrees and 37 degrees C. In contrast, the ligands whose Ki values are increased by muscimol either decreased or did not alter the Ki values at 37 degrees C as compared to those at 0 degree C. Incubation of membranes at 37 degrees C for 30 min accelerated gamma-aminobutyric acid (GABA) release by 221% over that at 0 degree C. These results indicate that changes in incubation temperature alter benzodiazepine receptor affinity for ligands via GABA.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in hypoxia

Exercise tolerance is impaired in hypoxia, and it has recently been shown that dietary nitrate supplementation can reduce the oxygen (O(2)) cost of muscle contractions. Therefore, we investigated the effect of dietary nitrate supplementation on arterial, muscle, and cerebral oxygenation status, symptoms of acute mountain sickness (AMS), and exercise tolerance at simulated 5,000 m altitude. Fifteen young, healthy volunteers participated in three experimental sessions according to a crossover study design. From 6 days prior to each session, subjects received either beetroot (BR) juice delivering 0.07 mmol nitrate/kg body wt/day or a control drink (CON). One session was in normoxia with CON (NOR(CON)); the two other sessions were in hypoxia (11% O(2)), with either CON (HYP(CON)) or BR (HYP(BR)). Subjects first cycled for 20 min at 45% of peak O(2) consumption (VO(2)peak; EX(45%)) and thereafter, performed a maximal incremental exercise test (EX(max)). Whole-body VO(2), arterial O(2) saturation (%SpO(2)) via pulsoximetry, and tissue oxygenation index of both muscle (TOI(M)) and cerebral (TOI(C)) tissue by near-infrared spectroscopy were measured. Hypoxia per se substantially reduced VO(2)peak, %SpO(2), TOI(M), and TOI(C) (NOR(CON) vs. HYP(CON), P < 0.05). Compared with HYP(CON), VO(2) at rest and during EX(45%) was lower in HYP(BR) (P < 0.05), whereas %SpO(2) was higher (P < 0.05). TOI(M) was ～4-5% higher in HYP(BR) than in HYP(CON) both at rest and during EX(45%) and EX(max) (P < 0.05). TOI(C) as well as the incidence of AMS symptoms were similar between HYP(CON) and HYP(BR) at any time. Hypoxia reduced time to exhaustion in EX(max) by 36% (P < 0.05), but this ergolytic effect was partly negated by BR (+5%, P < 0.05). Short-term dietary nitrate supplementation improves arterial and muscle oxygenation status but not cerebral oxygenation status during exercise in severe hypoxia. This is associated with improved exercise tolerance against the background of a similar incidence of AMS.     

Thyroid hormone response to thyrotropin releasing hormone after cold treatment during pre- and postnatal development in the domestic fowl

The purpose of the present study was to compare the effect of periodic cooling during the establishment of a functional pituitary-thyroid axis at days 11-14 of incubation and at other developmental stages, on the subsequent thyroid hormone response to thyrotropin releasing hormone (TRH). In the first and second experiment chick embryos were cooled for 6 hr/day to 30 degrees C from day 11 till 14 and from day 15 till 18 respectively, whereas control groups were incubated throughout at 37.8 degrees C. In both experiments the thyroxine (T4) response upon TRH in 19 day-old embryos was higher in the previously cold treated embryos, according to the percentages of increase. However, the higher T4 response in the cold treated animals disappeared in 1 or 7 day-old chicks hatched from the 2nd experiment, but remained present in chicks of the same ages in the 1st experiment. In a third experiment the T4 response to TRH injection immediately and 3 and 8 days after a temperature treatment (25 degrees C or 12 degrees C) for one week on four weeks old broiler chickens was found to be similar in both temperature groups. In all experiments there was a concomitant triiodothyronine (T3) increase after TRH injection, but differences between experimental groups were observed at days 15 and 19 of incubation and immediately after the postnatal temperature treatment. As an overall conclusion the results indicate that cold treatment only during the establishment of the hypothalamo-hypophysial control of thyroid function can have a long lasting effect by enhancing the T4 response to TRH injection.     

Social reinstatement responses of meat-type chickens to familiar and unfamiliar conspecifics after exposure to an acute stressor

Runway tests are considered indicative of underlying sociality in birds and their ability to make social discriminations. We evaluated whether experience of a prior stressor alters the subsequent affiliation responses of 9 or 10-day-old chicks simultaneously exposed to familiar (cagemates) and unfamiliar conspecifics placed in goal boxes at opposite ends of a runway. Birds were housed in groups of eight in home cages. Half of the birds in each home cage were used as either familiar or unfamiliar social stimuli in the goal boxes. The other half of the birds were randomly assigned either to a control (CON; n = 51) group that remained undisturbed until testing or to a stress-treatment (STR; n = 52) group that was exposed to a 5-min restraint stressor, returned to its home cage and then tested 1 h later. Birds were individually tested in the runway for 5 min and the behaviours video-recorded. During revision of tapes, the projected floor image of the runway was divided into squares and zones. The stressor decreased (P < 0.01) the time spent in close proximity (close zone; CZ) to conspecifics regardless of the familiarity of the stimulus birds. Regardless of treatment, test chicks showed shorter latencies to enter (P < 0.05) and spent longer time (P < 0.02) in the familiar than in the unfamiliar CZ suggesting that young chicks can discriminate between familiar and unfamiliar conspecifics encountered in novel surroundings. While in close proximity to familiar conspecifics, STR birds showed a reduced (P < 0.05) number of squares entered compared to CONs. This reduced locomotor activity was not accompanied by an increased activity in other zones of the runway. At the end of the trial, both CON and STR birds showed a reduced (P < 0.05) locomotor activity in the unfamiliar CZ and an increased (P < 0.05) activity in the central zone of the runway. Interestingly, no differences were detected between CON and STR birds in the total number of squares entered during the trial. These results suggest that prior stressor exposure did not affect the overall amount of locomotion but altered the spatial distribution of it. Collectively, our findings suggest that exposure to an acute stressor event subsequently affects chicks’ affiliation responses in runway tests. The way a bird will react depends on the identity (familiar or unfamiliar) of the conspecifics in its close environment.     

Elevation of body temperature is an essential factor for exercise-increased extracellular heat shock protein 72 level in rat plasma

This study examined whether the exercise-increased extracellular heat shock protein 72 (eHsp72) levels in rats was associated with body temperature elevation during exercise. In all, 26 female Sprague-Dawley rats (3 mo old) were assigned randomly to control (CON; n = 8), exercise under warm temperature (WEx; n = 9), or exercise under cold temperature (CEx; n = 9). The WEx and CEx were trained at 25 degrees C or 4 degrees C, respectively, for nine days using a treadmill. Before and immediately after the final exercise bout, the colonic temperatures were measured as an index of body temperature. The animals were subsequently anesthetized, and blood samples were collected and centrifuged. Plasma samples were obtained to assess their eHsp72 levels. Only the colonic temperature in WEx was increased significantly (P < 0.05) by exercise. The eHsp72 level in WEx was significantly higher (P < 0.05) than that of either the CON or CEx. However, no significant difference was found between CON and CEx. Regression analyses revealed that the eHsp72 level increased as a function of the body temperature. In another experiment, the eHsp72 level of animals with body temperature that was passively elevated through similar kinetics to those of the exercise was studied. Results of this experiment showed that mere body temperature elevation was insufficient to induce eHsp72 responses. Collectively, our results suggest that body temperature elevation during exercise is important for induction of exercise-increased eHsp72. In addition, the possible role of body temperature elevation is displayed when the exercise stressor is combined with it.     

Thermal instability of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase function

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca(2+)-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for < or =60 min either at a normal resting body temperature (37 degrees C) or at a temperature indicative of exercise-induced hyperthermia (41 degrees C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca(2+)-ATPase and Ca(2+) uptake values (nmol. mg protein(-1). min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca(2+) uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37 degrees C (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37 degrees C, ryanodine (300 microM), which was used to block Ca(2+) release from the calcium release channel, prevented the time-dependent decrease in Ca(2+) uptake. A general inactivation (P < 0.05) of maximal Ca(2+)-ATPase activity (V(max)) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca(2+) concentration required for half-maximal activation of Ca(2+)-ATPase activity, decreased (P < 0.05) at 41 degrees C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V(max) with the Ca(2+) ionophore A-23187 was less pronounced at 41 degrees C compared with 37 degrees C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca(2+) uptake and occurs as a result of increases in membrane permeability to Ca(2+). Moreover, the decreases in Ca(2+)-ATPase kinetics in WG with sustained heat stress result from SH oxidation.     

Benzodiazepine Binding to Cultured Human Pituitary Cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.     

Photoaffinity Labeling of Different Benzodiazepine Receptors at Physiological Temperature

Irreversible labeling of benzodiazepine receptors in membranes from cerebellum or hippocampus was compared at 0 degrees C using [3H]flunitrazepam as a photoaffinity ligand. [3H]Flunitrazepam reproducibly and irreversibly labeled mainly one protein (P51) in cerebellum and at least two proteins (P51 and P55) in hippocampus at both temperatures. Differential inhibition at 37 degrees C of irreversible [3H]flunitrazepam binding to the individual proteins by several selective benzodiazepine receptor ligands supports the hypothesis that P51 and P55 are associated with different benzodiazepine receptors.     

Temperature Dependence of [3H]Ro 15–1788 Binding to Benzodiazepine Receptors in Human Postmortem Brain Homogenates

The temperature dependence of in vitro binding of [3H]Ro 15-1788 to benzodiazepine receptors in human postmortem neocortex and neocerebellum homogenates was studied. An increase of the equilibrium dissociation constants (KD) from 1.40 nmol/L and 1.04 nmol/L at 4 degrees C to 6.10 nmol/L and 8.91 nmol/L at 37 degrees C was found for neocortex and neocerebellum, respectively. In contrast, maximal binding (Bmax) remained in the range of 30-35 fmol/mg for neocortex and 24-27 fmol/mg of tissue (wet weight) for neocerebellum at all the temperatures. The KD of 6.10 nmol/L for neocortex at 37 degrees C in vitro is of the same order as the KD of 10 nmol/L obtained by positron emission tomography for [11C]Ro 15-1788 binding to benzodiazepine receptors in the human neocortex in vivo. The differences in KD between in vitro and in vivo benzodiazepine receptor binding to human neocortex and cerebellum seem to be due at least partially to temperature differences of in vitro and in vivo studies.     

The benzodiazepine receptor ligand, methyl beta-carboline-3-carboxylate, is both sedative and proconvulsant in chicks

Certain pharmacological properties of methyl beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine receptor ligand, have been investigated in chicks. Although beta-CCM has been established previously as an "inverse agonist" of benzodiazepine receptors in rodents, having effects opposite to those of benzodiazepines in a variety of tests, in chicks this compound had a different pharmacological profile. Firstly, in contrast to the overt convulsant action of beta-CCM in other species, beta-CCM (0.05-40 mg/kg) did not produce convulsions by itself in chicks, but it was only proconvulsant. Secondly and most surprisingly, beta-CCM, like diazepam, produced in chicks a sedation which could be blocked by the benzodiazepine receptor antagonist Ro 15-1788. Thus it appears that beta-CCM can function both as an agonist and as an inverse agonist in this animal.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Postmortem Changes in Rat Brain: Studies on Membrane-Bound Enzymes and Receptors

The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4 degrees C and 25 degrees C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague-Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4 degrees C for up to 72 h prior to freezing at -70 degrees C. Na,K-ATPase and low-affinity Ca-ATPase activities were also stable in brains kept at 25 degrees C for up to 72 h. Mg-ATPase activity was reduced in brains kept at 25 degrees C for 24 and 48 h. [3H]Guanidinoethylmercaptosuccinic acid [( 3H]GEMSA) binding to enkephalin convertase in the cytosol was not significantly changed in brains kept at 25 degrees C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate [( 3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25 degrees C for 72 h. Opioid receptor binding also decreased in brains kept at 25 degrees C. Using [3H]2-D-alanine-5-D-leucine enkephalin to label delta opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25 degrees C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25 degrees C, but was decreased after 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.