L.D.H. and L.D.H. isoenzymes of the intra-uterine secretions of the cow during the estrous cycle

Uterine secretions were collected from 20 mature cows during estrus (day 0), metestrus (day 5), diestrus (day 10) and proestrus (day-1). Lactate dehydrogenase (LDH) and LDH isoenzymes activity were evaluated. No significant cyclic variations of LDH activity was found in the uterine secretions while the mean of the enzyme activity was higher during the estrogenic period of the cycle. The relative activity of LDH-1, LDH-2 and LDH-3 isoenzymes were higher during proestrus and estrus whereas LDH-5 activity was more important during metestrus. The LDH-3 seems to have the higher relative activity in uterine secretions of the cow.     

Isothiocyanates. A new class of uncouplers.

This paper describes the uncoupling effect of three isothiocyanates: p-bromophenylisothiocyanate, 4,4'-diisothiocyanatebiphenyl and beta-naphtylemthylisothiocyanate on the respiration of Ehrlich-Lettré cells and isolated mitochondria. The isothiocyanates are similar to other uncouplers (such as 2,4-dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone) in that they: 1. stimulate respiration of state 4 mitochondria; 2. stimulate mitochondrial ATPase activity; 3. release the inhibition of mitochondrial respiration by oligomycin and 4. inhibit both mitochondrial respiration and mitochondrial ATPase activity at higher molar concentrations. The incoupling activity of these isothiocyanates correlates well with their biological activity. Maximal activation of a latent mitochondrial ATPase activity of rat liver mitochondria in the presence of p-bromophenylisothiocyanate was found at a concentration of 15 muM. The investigated isothiocyanates differ significantly in their solubility in organic solvents and their chemical reactivity. We assume that the greater the partition coefficient in a series of isothiocyanates grouped according to the increasing value of log P (partition coefficient for the system octanol/water, 25 degrees C), the greater will be their uncoupling activity, but only up to a certain degree. Any further increase of log P will be marked by a decrease of this activity.     

Beta-glycosidases and diabetic microangiopathy. II. An insulin-dependent isozyme of beta-N-acetylglucosaminidase.

Previously we reported that beta-glycosidase activities were markedly decreased in the kidney but increased in the serum of diabetic rats. To examine these changes, the isozymes of beta-N-acetylglucosaminidase [EC 3.2.1.30] of rats were examined by DEAE-cellulose column chromatography. At least 3 major isozymes were found in both the kidney and liver. The main isozyme was type II isozyme in normal rat kidney and type III in normal rat liver. The activity of the type II isozyme in the kidney was markedly lowered when the total activity was decreased in diabetes and its normal activity was restored on insulin treatment, in parallel with increase in the total activity in diabetes. No significant change was found in the chromatographic pattern of isozymes in the liver in diabetes. In diabetic rat serum, the increase of total activity was found to be due to increase of type I and II isozymes.     

Studies on the circadian rhythm of phosphoenolpyruvate carboxykinase. III. Circadian rhythm in the kidney.

Phosphoenolypyruvate carboxykinase [EC 4.1.1.21] activity in rat kidney shows a circadian rhythm with the highest activity between 0200 h and 0800 h and the lowest activity between 1400 h and 2000 h. The rhythm was observed in both sexes and throughout the year. Actinomycin D and cycloheximide effectively blocked the circadian increase in enzyme activity. These findings suggest that the circadian increase in phosphoenolypyruvate carboxykinase activity is due to net synthesis of enzyme protein through newly synthesized mRNA. In experiments with kidney cortex slices, gluconeogenesis from the radioactive precursor, [14C]malic acid, was considerably higher at 0200 h than at 1400 h, varying in parallel with the change in the enzyme activity.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

Polycations as prostaglandin sythesis inducers. II. structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (>80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. Full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Sites of acid phosphatase in the differentiating root protophloem of Nymphoides peltata (S.G. Gmel.) O. Kuntze.

Abstract Sites of acid-phosphatase activity were found in the differentiating root protophloem of Nymphoides peltata by lead-salt and by azo-dye methods. Different substrates revealed different subcellular locations of the enzyme. The substrates β-glycerophosphate (β-GP) and naphthol ASBI phosphate revealed enzyme activity at similar sites within the sieve element. These sites included plasmodesmata, dictyosomes and small vacuoles in the cytoplasm. The substrate p-nitrophenylphosphate (p-NPP), however, revealed additional sites of acid-phosphatase activity which were not detectable by either naphthol ASBI phosphate or β-GP. For example, the inner region of the wall in mature sieve elements showed conspicuous acid-phosphatase activity only when p-NPP was used as substrate. The significance of the different locations of acid phosphatase within the sieve element is discussed. The convoluted ER, characteristic of immature sieve elements of N. peltata, failed to show acid-phosphatase activity whichever substate was used. By contrast, the stacked ER found in the parietal layer of mature sieve elements showed prominent acid-phosphatase activity regardless of the substrate used. The demonstration of acid-phosphatase activity in the stacked ER, and by both lead-salt and azo-dye methods, suggests that this organelle is a true site of acid-phosphatase activity. The onset of acid-phosphatase activity in the ER in later stages of sieve-element differentiation is compatible with the view that stacked ER plays a role in the final autolysis of the sieve-element protoplast.     

Studies on Amino Acids. XI.

As a part of the studies intending to clarify biological significance of the presence of acylase, enzymatic activity which hydrolyzes acyl amino acids, its activity in yeast was investigated. As a results, the occurrence in yeast of acylase activity was confirmed for the first time.

In order to study the enzymatic properties of this acylase activity, experiments were carried out with the enzyme preparation from brewer’s yeast. As a result of the investigation, yeast acylase was found to be able to hydrolyze a number of acyl amino acids, of these chloroacetyl derivatives especially readily, as in the case of previously studied acylase activity in other sources such as mold acylase. Several observations on the influence of metal ions and inhibitors, optical specificity and others were also presented.     

Lipoprotein lipase in rat lung. Effect of dexamethasone.

The effect of hormone administration on the activity of lipoprotein lipase in the lung was studied in the rat. The following hormones were administered: dexamethasone, L-thyroxine, estradiol-17beta and progesterone. In addition, lung lipoprotein lipase activity was studied in diabetic and lactating rats. Lipoprotein lipase activity was measured in dried, defatted preparations of rat lung using double labeled ([14C]palmitate, [3H]glycerol) chylomicron triacylglycerol as substrate. Dexamethasone administration caused a rise of 70% in the level of activity of lipoprotein lipase in acetone powders of lung and a 100% increase in the amount of enzyme released during heparin infusion into isolated, perfused lungs. Enzyme activity was higher in lungs of females than of male rats; however; the level of activity was unaffected by estrogen or progesterone administration to either male or ovariectomized rats. Diabetes, hyperthyroidism or lactation did not change lipoprotein lipase activity in the lung. The constant presence of lipoprotein lipase activity in the lung suggests that this organ is able to maintain a steady supply of triacylglycerol-fatty acids under a variety of physiological and pathological conditions. Stimulation of enzyme activity by dexamethasone could lead to increased uptake of triacylglycerol-fatty acids by the lung and may thus be a contributing factor to corticosteroid-induced enhanced surfactant synthesis.     

Amine oxidase-like activity of polyphenols. Mechanism and properties.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.     

Polycations as prostaglandin synthesis inducers. II. Structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (greater than 80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Studies on aspartase. IV. Reversible denaturation of Escherichia coli aspartase.

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity. While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity. Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly. Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions. Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation. In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted. Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min. Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules. These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity.     

Lipid metabolism by The gall-bladder. II. The in vitro conversion of lysophosphatidylcholine to phosphatidylcholine.

Lysophosphatidylcholine acyltransferase, which catalyzes the acylation of lysophosphatidylcholine with fatty acid coenzyme A to form phosphatidylcholine, was assayed in gall-bladder mucosa. In guinea pig gall-bladder the activity parallels that of the microsomal enzyme, glucose-6-phosphatase with 3--4-fold enrichment of the activity in the microsomes. Studies with saturated and unsaturated substrates demonstrated highest activity when oleoyl coenzyme A and palmitoyl lysophosphatidylcholine were used and the lowest activity when palmitoyl coenzyme A and palmitoyl lysophosphatidylcholine were used. This activity was demonstrated in the dog, rabbit, cat, calf and human gall-bladder mucosa; however, a wide variation in the amount was observed. Lysophospholipase, which catalyzes the hydrolysis of lysophosphatidylcholine to glycerophosphorylcholine and fatty acid, was also demonstrated in gall-bladder mucosa.     

Luteolytic prostaglandins. Synthesis and biological activity.

Analogues of PGF2 alpha with enhanced luteolytic activity were synthesized using the Corey synthesis. The luteolytic activity of the new prostaglandins was tested in the hamster. In addition the smooth muscle activity of the new compounds was compared with that of PGA2 on the longitudinal strip of rat stomach fundus. Structure-activity relationships in the new series of 17,18,19,20-tetranor-16-thienyl-oxy-PGF2 alpha are discussed.     

Sedating effects of Humulus lupulus L. extracts.

It was the aim of the study to check ethanolic and CO2 extracts from Humulus lupulus for sedating activity. Both preparations reduced the spontaneous locomotor activity, increased the ketamine-induced sleeping time and reduced body temperature, confirming a central sedating effect. No indications of anxiolytic activity were found in the elevated plus maze test for any of the test preparations. This sedating activity could be attributed to three categories of constituents of lipophilic hops extracts. Though the alpha-bitter acids proved to the be most active constituents, the beta-bitter acids and the hop oil clearly contributed to the sedating activity of lipophilic Humulus extracts.     

Regeneration of masseter muscle following lidocaine-induced degeneration. A histochemical study.

The histoenzymatic characteristics of regenerating myofibers of rat masseter muscle following injection of 1% lidocaine, as well as morphometric and histochemical characteristics of the typical myofibers, were investigated. Myoblasts appeared initially by day 1 among numerous macrophages within the confines of degenerating myofibers. Myotubes predominated by the 3rd day. Complete regeneration of the muscle occurred by at least 45 days. Phosphorylase activity was absent at day 1 and reappeared by the 5th day when the regenerating myofibers showed slight activity. By the 15th day the myofiber types had partly differentiated; red myofibers were smaller and stained less intensely than the white myofibers. Myotubes stained uniformly for succinic dehydrogenase activity from 3 until 5 days. After 5 days this staining increased gradually. Myofiber types began differentiation by 15 days and were fully differentiated by 45 days. ATPase activity was barely evident by 1-3 days. This activity appeared uniformly low up to 5 days and increased to an intensity comparable with that of the typical myofiber by 15 days. Slight leucine aminopeptidase activity occurred in macrophages 1 day following injection. By 3 days this activity appeared in the remaining myoblasts and in the myotubes. Some activity was found in the fibroblasts. This staining intensity at 5 days was equal to that of earlier lesions. A trace of this activity was found at 7 days, and none at 15 days. Glucose-6-phosphate dehydrogenase activity was present in the macrophages by day 1. It increased by 3 days and occurred mainly in myoblasts and myotubes. This activity decreased by 5 days, and none was found by 7 days.     

Lymphocyte plasma membranes. V. Specificity and mitogenicity of absorbed anti-lymphocyte sera.

Anti-lymphocyte sera against human thymus [ALS-(THY)] were absorbed serially with cultured human lymphoblasts (CHL) or thymus and residual antigen-binding activity was tested. The absorbed ALS were used to bind 125I-labeled antigens from lymphocytes labeled by the lactoperoxidase catalyzed iodination technique. Absorption of ALS(THY) with CHL led to the absorbed serum having less than 5 to 10% of its original antigen-binding activity against labeled CHL antigens while maintaining from 20 to 40% of its original activity against labeled THY. Serial absorption of ALS(THY) with THY led to an equal decrease in activity against both THY and CHI. When the immunoprecipitates from these experiments were examined on polyacrylamide gels containing SDS it was found that serial absorption of ALS(THY) with THY first removed activity against a component of m.w. similar to 48,000 leaving relatively greater activity against material of apparent high molecular wieght. In contrast, absorption of ALS-(THY) with CHL removed the antibodies against the high molecular weight material while leaving activity against the component of m.w. 48,000. When these absorbed ALS were used to induce in vitro lymphocyte proliferation, it was found that ALS(THY) absorbed with CHL, did not. The retention or loss of mitogenicity seemed to correlate with retention or loss of binding activity against the component(s) of m.w. similar to 48,000.     

Studies on new intracellular proteases in various organs of rat. 3. Control of group-specific protease under physiological conditions.

1. To study the role of group-specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined. 2. Studies on the distribution of group-specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine. 3. Group-specific protease in skeletal muscle increased under various dietary conditions (starvation, protein-free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group-specific protease in liver. 4. The level of group-specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease. 5. Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanism for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.     

Genetics of esterases inDrosophila. IX. Characterization of the JH-esterase inD. virilis

The kinetic characteristics of the main isozymes of Drosophila virilis esterase were studied and Km values of esterase-2, -4, and -6 and p-esterase for alpha- and beta-naphthyl acetate were obtained. Juvenile hormone (JH) was shown to inhibit the p-esterase activity when in competition with beta-naphthyl acetate and the general esterase inhibitor, diisopropylphosphofluoridate (DFP), was shown to inhibit all the components of the D. virilis esterase patterns except p-esterase. While studying the changes of p-esterase activity in D. virilis ontogenesis, the increase in p-esterase activity in the wandering larvae, prepupae, and early pupae was found to correlate with a decrease in JH titer at these stages. The decrease in JH level in a temperature-sensitive lethal mutant larvae of D. virilis at high temperatures was shown to correlate with increased p-esterase activity. These results confirm that p-esterase of D. virilis is JH-esterase.