Regulation of AMP-deaminase activity from white muscle of common carp Cyprinus carpio

AMP-deaminase was purified to electrophoretic homogeneity from white skeletal muscle of a teleost fish, the common carp, Cyprinus carpio. The purified enzyme was highly stable and showed non-Michaelis-Menten kinetics with a S(0.5) value for AMP of 2.52+/-0.16 mM (SEM) and a Hill coefficient of 1.19+/-0.11. Specific activity of the purified enzyme was 1000-1200 U/mg protein. The pH optimum was 6.3 and the enzyme was activated by ADP and ATP, but inhibited by phosphate and fluoride. Low concentrations of NaCl and KCl (100-150 mM) activated, whereas higher concentrations were inhibitory. Free radicals inactivated the enzyme, decreasing V(max) by one-half but not affecting S(0.5) or Hill coefficient. Possible regulatory mechanisms of AMP-deaminase activity in fish muscle are discussed.     

Digestive enzyme patterns and evaluation of protease classes in Catla catla (family: Cyprinidae) during early developmental stages

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12+/-0.01 mg maltose mg protein(-1) h(-1) in fish 4 days after hatching (DAH) and reached a maximum on (0.41+/-0.12 mg maltose mg protein(-1) h(-1)) 34 DAH. Total protease activity was minimum (123.2+/-16.5 mU mg protein(-1) min(-1)) on day-8 and reached its highest level (2713+/-147.2 mU mg protein(-1) min(-1)) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1+/-7.09 mU mg protein(-1) min(-1)). Highest chymotrypsin activity was found on day-32 (1789.0+/-111.7 mU mg protein(-1) min(-1)). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8-127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8-37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5+/-1.19% to 92.8+/-0.85%, 53.3+/-9.47% to 90.5+/-2.6% and 39.8+/-3.8% to 84.7+/-1.54% of total protease activity, respectively. There was only 2.58+/-0.66% to 10.21+/-0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.     

A comparison of the non-specific acid phosphomonoesterase activity in the larva of Phocanema decipiens (Nematoda) with that of the muscle of its host the codfish (Gadus morhua).

The non-specific phosphomonoesterase (enzyme I) extracted from the larva of the codworm (Phocanema decipiens) is different from the enzyme (enzyme II) from the muscle of its host, the codfish (Gadus morhua). The pH optima were 4.0 and 4.5, and the KM values for p-nitrophenyl phosphate hydrolysis were 1.8 mM and 6.5 mM for enzymes I and II respectively. The specific specific activity in units (0.01 mumol/min) per mg protein was 4.80 +/- 0.85 and 0.54 +/- 0.07 for enzymes I and II respectively. The specific activity from uninfected muscles was only 0.39 (SD +/- 0.017) units per mg of protein. Both enzymes were inhibited by NaF, HgCl2, and cysteine but were stimulated by 2-mercaptoethanol. EDTA and iodoacetamide had no effect on enzyme I but enzyme II was activated by EDTA and inhibited by iodoacetamide. Cadmium ions inhibited both the enzymes but a conspicuous feature with enzyme II was in the increase in percentage inhibition by lowering the concentration of CD2+.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitors of phosphodiesterase III block stimulation of Xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 +/- 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 +/- 1 microM papaverine, 20 +/- 2 microM isobutylmethylxanthine, and 128 +/- 16 microM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 +/- 0.3 microM Cl-930 and 84 +/- 23 microM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone-stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.     

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle.

Many cyprinid fish are able to compensate for a decrease in ambient temperature by process of physiological adaptation in the function of muscles. In the winter habitat of crucian carp (Carassius carassius L.), low temperature is associated with simultaneous oxygen shortage. Because of the oxygen deprivation, there is probably little space for compensatory adaptation because positive thermal compensation would increase energy demand and accelerate depletion of glycogen reserves. Thus, we assumed that the crucian carp, unlike many other cyprinid fish, would not show positive thermal compensation but either no compensation or inverse compensation in muscle function. To test this hypothesis in the relaxation system of skeletal muscles, we determined the parvalbumin content and the activity of sarcoplasmic reticular (SR) Ca-ATPase in white myotomal muscle of winter- and summer-acclimated crucian carp. In the laboratory, the winter fish were kept at 2 degrees C and the summer fish at 22 degrees C for a minimum of 3 weeks before the experiments. The specific activity of SR Ca-ATPase at low experimental temperature (2 degrees C) was similar in summer- and winter-acclimated fish (0.26 +/- 0.04 vs. 0.25 +/- 0.04 mM/mg/min; P > 0.05). Because of the bigger Q(10) of cold-acclimated carp, the enzyme activity at 30 degrees C was higher in cold-acclimated winter fish than in warm-acclimated summer fish (7.42 +/- 0.90 vs. 5.18 +/- 0.53 mM/mg/min; P < 0.05). In contrast, the yield of SR protein was 70% higher in summer than winter fish (0.315 +/- 0.045 vs. 0.187 +/- 0.017 mg/g; P < 0.001). Because of these opposing changes, total Ca-ATPase activity of SR (per gram muscle weight) remained relatively constant. Similarly, the parvalbumin content of the myotomal muscle was not different between summer (4.09 +/- 0.95 mg/g) and winter (3.70 +/- 0.60 mg/g) fish. Although there were no seasonal changes in the total relaxing system of the crucian carp white myotomal muscle, the same activity of SR Ca-ATPase in winter fish was obtained with less amount of SR pump protein, owing to the increased catalytic activity of the enzyme. The higher catalytic activity of winter fish SR Ca-ATPase might be caused by differences in fatty acid composition noted in membrane lipids; i.e., fewer saturated fatty acids and more n-6 polyunsaturated fatty acids (PUFAs), at the expense of n-3 PUFAs, were present in the SR of cold-acclimated winter fish. Temperature-induced changes in enzyme protein, however, cannot be excluded. Thus, the present results indicate the absence of positive thermal compensation in the relaxing system of crucian carp white muscle. It seems, however, that lipid composition of SR membranes and temperature dependence of SR Ca-ATPase are altered by seasonal acclimation.     

Purification and characterization of an arylamine N-acetyltransferase in the nematode Enterobius vermicularis.

N-acetyltransferase (NAT) activities were determined by incubation of Enterobius vermicularis cytosols with 2-aminofluorene (2-AF) as the substrate followed by high pressure liquid chromatography assays. The NAT activity from E. vermicularis was found to be 0.41 +/- 0.08 nmol/min/mg protein for 2-AF. The apparent K(m) and Vmax values obtained were 0.81 +/- 0.11 mM and 2.25 +/- 0.22 nmol/min/mg protein respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for 2-AF. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. vermicularis was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were the most potent inhibitors. Of the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetate, in contrast to other agents, markedly inhibited NAT activity. This report is the first demonstration of acetyl coenzyme A-dependent arylamine NAT activity in E. vermicularis and extends the number of phyla in which this activity has been found.     

Improved determination of bovine glutaminyl cyclase activity using precolumn derivatization and reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of l-pyroglutamic acid beta-naphthylamide at 235 nm, enzymatically formed from the substrate l-glutaminyl-beta-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0-8.5. The K(m) and V(max) values were 100.2+/-2.9 microM and 332 +/-21.7 pmol/(h microg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Glutamate:glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.)

Glutamate:glyoxylate aminotransferase from green parts of 7-day-old rye seedlings was purified almost to homogeneity. Specific activity of the purified enzyme measured with L-glutamate and glyoxylate as substrates, was 46.1 units/mg. The enzyme activity with L-alanine and 2-oxoglutarate as substrates was higher by a factor of 1.5, whereas with L-alanine and glyoxylate or L-glutamate and pyruvate it was similar to that with L-glutamate and glyoxylate. L-Aspartate, L-arginine and L-ornithine could also serve as substrate. The reaction followed the Ping-Pong Bi Bi mechanism and Km values for L-glutamate and glyoxylate were 2.6 and 0.5 mM, respectively. Pyridoxal phosphate was found to be the coenzyme of glutamate-glyoxylate aminotransferase. This coenzyme was rather tightly bound with the enzyme protein, as the attempts at its complete resolution from the apoenzyme were unsuccessful. Pyridoxal phosphate, 2-mercaptoethanol and sucrose, or bovine serum albumin stabilized the enzyme. Molecular weight of glutamate:glyoxylate aminotransferase from rye seedlings, determined by SDS-polyacrylamide gel electrophoresis, was 58,800 +/- 2,100, whereas molecular sieving on Sephacryl S-200 gel gave values of 70,800 +/- 700 or 61,400. Similar values obtained for the denatured and nondenatured enzyme seem to indicate that it is a monomeric protein.     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

Comparison of nuclear 5 alpha-reductase activities in the stromal and epithelial fractions of human prostatic tissue

The nuclear conversion of testosterone (T) to dihydrotestosterone (DHT) was compared in the separated stromal and epithelial fractions of hyperplastic (n = 20), malignant (n = 5) and normal (n = 1) prostatic tissues. Standard assay conditions were: 1 microM testosterone, plus 4-6 X 10(5) DPM [3H]T, 1.0 mM NADPH, 2.0 mM EDTA and 0.5-1.0 mg nuclear protein in a total volume of 1.1 ml HEPES buffer, pH 7.4 (stroma) or MES buffer, pH 6.5 (epithelium). The apparent Km values for the stromal enzyme were 0.2, 0.2 and 0.3 microM, respectively, for the enzymes in hyperplastic, malignant and normal tissues. The Vmax values were 26 +/- 4.2, 2.8 +/- 0.6 and 4.1 pmol/mg protein/30 min incubation, respectively, for these same tissues. The apparent Km values for the epithelial enzymes, from the same tissues, were 0.03, 0.07 and 0.08 microM. The Vmax values for the epithelial enzymes were 4.8 +/- 1.2, 0.69 +/- 0.08 and 1.1 pmol/mg protein/30 min incubation. The pH optimum for the stromal enzyme lay between pH 6.5 and 7.5, whereas the pH optimum for the epithelial enzyme lay between 5.5 and 6.5. Enzymatic activity in both fractions revealed a biphasic response to zinc. In the absence of EDTA, microM quantities of zinc enhanced enzymatic activity while mM quantities inhibited this activity. These results would suggest that differences in the conversion of T to DHT help to explain, at least in part, the higher DHT levels seen in hyperplastic tissue and the higher T levels seen in the malignant prostate.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (Cyprinus carpio L.)

The acclimation temperature of carp does not affect the amount of cytochrome c oxidase per mg mitochondrial protein as revealed from the reduced-minus-oxidized difference spectra of red muscle mitochondria from cold- and warm-acclimated carp. There are no differences between cold- and warm-acclimated fish in the substrate binding properties of the enzyme as judged from the Km values for cytochrome c at 30 degrees C (3.34 +/- 0.ee microM, acclimation temperature 10 degrees C and 3.55 +/- 0.31 microM, acclimation temperature 30 degrees C). The molar activities of the enzyme, however, differ for both acclimation temperatures: when intercalated in the 10 degrees C-acclimated mitochondrial membrane, the enzyme can catalyze the oxidation of 117.6 +/- 17.2 mol ferrocytochrome c/s per mol heme a as compared with 85.6 +/- 17.2 in the 30 degrees C-acclimated membrane (experimental temperature 30 degrees C). Correspondingly, higher specific activities of the succinate oxidase system are observed in mitochondria from cold-acclimated carp as compared with those obtained from warm-acclimated carp. The results indicate that cold acclimation of the eurythermic carp is accompanied by a partial compensation of the acute effect of decreasing temperature on the activity of cytochrome c oxidase in red muscle mitochondria. Based on the temperature-induced lipid adaptation reported for carp red muscle mitochondria (Wodtke, E. (1980) Biochim. Biophys. Acta 640, 698--709), it is concluded that during thermal acclimation the molar activity of cytochrome c oxidase is controlled by viscotropic regulation. The results fit to the conception that cardiolipin constitutes a lipid shell (annulus) surrounding the oxidase within the native membrane, but that it is the bilayer fluidity and not the annular fluidity which determines the activity of cytochrome c oxidase.     

Ca2+/protein modulator-dependent and -independent cyclic GMP phosphodiesterase from hog heart.

Ca2+/protein modulator-dependent and -independent guanosine 3':5'-monophosphate (cGMP) phosphodiesterases were separated from hog heart. The protein modulator-free Ca2+/protein modulator-dependent enzyme was partially purified by repeated DEAE-cellulose column chromatography and heat treatment. The final preparation of this enzyme showed no significant basal activity under the standard assay conditions. Lineweaver-Burk plots of the Ca2+/protein modulator-dependent enzyme activity indicated the presence of only a single kinetic form of the enzyme with Km=2.0 X 10(-6) M for for cGMP, whereas the plots for the independent enzyme were anomalous, showing both high and low K m values for cGMP. The Ca2+/protein modulator-dependent enzyme proved relatively stable at 48 degrees C for 1 h, but the independent form lost its activity under the same conditions. Furthermore, 50% inhibition of the dependent enzyme activity, but only 10% inhibition of the independent enzyme activity, was observed with 0.1 mM adenosine 3':5'-monophosphate (cAMP) when 1 muM cGMP was employed as a substrate.