Effect of chloroform and ether narcosis on the activity of basic carboxypeptidases in the hypothalamic-pituitary-adrenal axis in rats

It is discovered that chloroform narcosis does not influence on carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity in the rats hypothalamic-pituitary-adrenal axis. Ether narcosis provokes an increase of PMSF-inhibited carboxypeptidase activity in the pituitary body approximately in 8 times and carboxypeptidase H activity in hypothalamus by 29 percents in comparison with the intact animals. It is supposed that at research neuropeptides and their metabolism enzymes and especially the answer to a stress chloroform narcosis would be the better anaesthesia method than ether narcosis.     

Activity of the basic carboxypeptidases in female rat tissues at different stages of estrus cycle

It is revealed, that the activity of neuropeptide metabolism enzymes (carboxypeptidase H, phenylmethylsulfonilfluorid-inhibited carboxypeptidase) in the female rat tissues depends upon the stage of estrus cycle. The carboxypeptidase H activity in the pituitary gland is the highest in proestrus; it is almost 3 times higher in comparison with diestrus; it is a little bit higher in striatum on the stage of estrus, than in diestrus and proestrus, in adrenals on the stage of proestrus and estrus it is a little bit lower, than in diestrus; in the ovaries on the stage of proestrus it is much higher, than in estrus and diestrus. The activity of PMSF-inhibited carboxypeptidase in ovaries on the stage of proestrus and diestrus is 1.7-1.8 times higher, than at the stage of diestrus. The activity of carboxypeptidase M in adrenal tissue at the stage of proestrus is 35-40% of that at the stage of diestrus and estrus. The activity of carboxypeptidase M in the ovaries at the stage of diestrus is 45-50% of that at the stage of diestrus and estrus. The role of the investigated enzymes in cyclic changes of a level of biologically active peptides and in regulation of estrus cycle is discussed.     

The effect of ethanol consumption by dams on the offspring locomotion in the open field test and carboxypeptidase activities in the rat brain and adrenal medulla

Consumption of dams ethanol increased the posterity locomotion activity in open field test. The increase in female rats was higher then in male ones. Differences in the carboxypeptidase H and PMSF-inhibited carboxypeptidase activities between the brain regions and adrenal medulla of prenatally exposed to ethanol and intact rats were found. The changing of enzyme activities in female rats was higher then in male ones. It is possible that dams ethanol consumption induced profound changes in locomotion mediated, at least partially, by changes in the rate of proteolytic processing of neuropeptide precursors.     

Isolation,partial purification,characterization, and tissue distribution of phenylmethylsulfonyl fluoride-inhibited feline carboxypeptidase

Phenylmethylsulfonyl fluoride (PMSF)-inhibited carboxypeptidase from cat liver was purified 148-fold by chromatography on CM- and DEAE-cellulose with 27.3% yield. Molecular weight of the enzyme is 100-110 kD as determined by gel filtration on Sephadex G-150. The enzyme has maximum activity at pH 5.50-5.75; its activity is completely inhibited by PMSF or p-chloromercuribenzoate and partially inhibited by iodoacetamide. EDTA, 2-mercaptoethanol, N-ethylmaleimide, Co²⁺ and Ca²⁺, basic carboxypeptidase inhibitor guanidinoethylmercaptosuccinic acid, and angiotensin-converting enzyme inhibitor captopril do not influence its activity. The enzyme cleaves arginine from enkephalin-Leu5-Arg6 and dansyl-Phe-Leu-Arg to form enkephalin-Leu5 and dansyl-Phe-Leu, respectively, and very slowly cleaves leucine from carbobenzoxy-Gly-Leu. Further cleavage of either enkephalin-Leu5 or dansyl-Phe-Leu was not detected. The highest activity of this enzyme was found in adrenal glands and testicles; this activity was 30% lower in hypophysis, and still lower in liver and kidney. The PMSF-inhibited carboxypeptidase activity in brain was about 6-16 times lower than that in adrenal gland. In brain regions, the highest activity was detected in gray matter of cerebral hemispheres and cerebellum, and slightly lower activity was found in thalamus/hypothalamus, striatum, and hippocampus. The lowest activity was found in quadrigeminal bodies, medulla oblongata, and white matter of cerebral hemispheres. The enzyme exists mainly in soluble form; the activity of membrane-associated enzyme is 7-25% of soluble enzyme activity depending on tissue type. We consider here a possible involvement of PMSF-inhibited carboxypeptidase in the metabolism of biologically active peptides.     

Carboxypeptidase H-like immunoreactivity in the striatum of cats and monkeys

The enzyme carboxypeptidase H was detected by immunohistochemistry in the striatum of adult cats and monkeys. Specific labelling was observed in the neuropil as well as in both medium-sized and large neuronal cell bodies. The distribution of neurons and neuropil expressing immunoreactivity to carboxypeptidase H was examined in relation to the pattern of immunoreactivity to the neuropeptides enkephalin and substance P. Carboxypeptidase H-like immunoreactivity was found both in zones rich and poor in immunostaining for the two peptides, but was usually denser in those striatal areas in which substance P-positive cell bodies are clustered (striosomes). The results further suggest a role for carboxypeptidase H in the metabolism of multiple neuropeptides in vivo.     

The effect of prenatal stress on the carboxypeptidase H activity in the hypothalamo-pituitary-adrenal-gonadal system of the rat

Effects of dams stress on the carboxypeptidase H: the neuropeptide exchange enzyme in the hypothalamo-pituitary-adrenall-gonadal system, was studied in the litter of different age (0, 14, 28, 45, and 120 day after birth) was studied. The sex differences in dynamic of enzyme activity in intact animals are substantiated. The effect of prenatal stress on carboxypeptidase H activity dependes on age and sex of animals. Prenatal stress is altering during the age dynamics of enzyme activity. This in the pituitary gland and hypothalamus of prenatal stressed female rats was a control for the male rats. In the adrenal and gonadal gland of prenatal stressed males, this was a control for the female rats female. The role of carboxypeptidase H in pubescence and mechanisms of effect of prenatal stress on sex system functional are discussed.     

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.     

The Physiological Significance of Mitochondrial Proton Leak in Animal Cells and Tissues

Mitochondrial proton leak is an important component of cellular metabolism in animals and may account for as much as one quarter to one third of the Standard Metabolic Rate of the rat. The activity of the proton leak pathway is different in a wide range of animal species and in different thyroid states. Such differences imply some function for proton leak and candidates for this function include thermogenesis, protection against reactive oxygen species, endowment of metabolic sensitivity and maintenance of carbon fluxes.     

Effect of testosterone on carboxypeptidase H activity in the murine hypothalamus and hypophysis

Effects of single intraperitoneal administration of testosterone propionate (3 or 30 mg/kg body weight) on activity of carboxypeptidase H in pituitary body and hypothalamus of female white mouse were studied. It was found that testosterone propionate treatment (3 and 30 mg/kg body weight) increased the carboxypeptidase H activity in pituitary through 0.5 hour and it decreased one in 24 hours after treatment. The carboxypeptidase H activity in hypothalamus was lower as compared with control animals in 24 hours after testosterone propionate treatment in the dose 3 mg/kg body weight. However, the carboxypeptidase H activity in hypothalamus was lower in 0.5 and 24 hours and it was higher in 4 h after testosterone propionate treatment in the dose 30 mg/kg body weight as compared with the control. These data suggest that testosterone affects the carboxypeptidase H activity by changing the level of enzyme gene expression.     

Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B

Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.     

Effect of stress factors on carboxypeptidase H activity in areas of the rat brain]

It is established that carboxypeptidase H activity increases as affected by various stress factors in the rat brain departments. The increase of the enzyme activity because of the emotional-pain stress is of continuous character. Possible role of carboxypeptidase H in the development of the stress response is discussed.     

Placental enzymes involved in regulatory peptide metabolism in EPH-gestosis

Essential edema-proteinuria-hypertension (EPH) gestosis still represents an important obstetrical problem. We have investigated the activity of carboxypeptidase H (CPH), phenylmethylsulfonyl fluoride inhibited carboxypeptidase (PMSF-CP), carboxypeptidase M (CPM) and angiotensin-converting enzyme (ACE), the main carboxypeptidases in human placenta under normal conditions and mild EPH-gestosis. Gestosis was accompanied by the decrease in activity of the enzymes involved into metabolism of regulatory peptides (ACE, CPH, PMSF-CP, CPM) compared with their activity in placenta under physiological pregnancy. Correlation analysis revealed positive correlation between placental CPH and CPM (r = 0.2735*) in EPH-gestosis. These findings suggest involvement of placental proteases into formation of compensatory-adaptive reactions in the fetoplacental complex at EPH-gestosis; the data obtained may be also employed for the development of methods of prophylaxis and corrections of metabolic impairments in pathology of pregnancy.     

Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation

Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors. Ecto-5′-nucleotidase (E5′N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.     

Specificity of the carboxypeptidase inhibitor from potatoes

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K_i about 10 micromolar) for a carboxypeptidase G₁ from an acinetobacterium was found in this study.     

Differences in Metabolism of Chemical Carcinogens in Cultured Human Epithelial Tissues and Cells

The metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen-DNA adducts are generally qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in out-bred animal species, including humans. These interindividual differences in amounts of carcinogen-DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10-to 150-fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.     

Rat preprocarboxypeptidase H. Cloning, characterization, and sequence of the cDNA and regulation of the mRNA by corticotropin-releasing factor

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of approximately 2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by approximately 30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.     

The subcellular localization of carboxypeptidase N in the substantia grisea of the cat brain]

The activity of carboxypeptidase H in the cat grey matter is established to be connected with microsomes. A possibility to involve carboxypeptidase H to the processing of neuropeptide predecessors in the encephalon is discussed.     

Longitudinal distribution and ecophysiological characteristics of Hydropsyche exocellata (Trichoptera: Hydropsychidae) in large rivers

The position as a potamic or rhithronic species of Hydropsyche exocellata (Trichoptera Hydropsychmae) was analysed with regard to 1) its metabolism temperature curve and the total amplitude of its respiratory metabolism between 5° and 25°C, n) its distribution in some reaches of the Rhône River basin (France), and Hydropsychmae coexisting species in those reaches to assess its place in the stream continuum Metabolism data indicated clearly that H exocellata was a species adapted to the potamon, but distribution data did not confirm metabolism results The absence of H exocellata, for instance, from the lower and potamic reaches from the Rhône River itself, as well as its frequent coexistence with H pellucidula, a well-established near-rhithronic species regarding metabolism, suggests a more complex definition of its position as a potamic species     

Measurement of carnitine biosynthesis enzyme activities by tandem mass spectrometry: differences between the mouse and the rat

Although the mouse frequently is used to study metabolism and deficiencies therein, little is known about carnitine biosynthesis in this animal. To this point, only laborious procedures have been described to measure the activity of carnitine biosynthesis enzymes using subcellular fractions as the enzyme source. We developed two simple tandem mass spectrometry-based methods to determine the activity of three carnitine biosynthesis enzymes (6-N-trimethyllysine dioxygenase, 4-trimethylaminobutyraldehyde dehydrogenase, and 4-trimethylaminobutyric acid dioxygenase) in total homogenates that can be prepared from frozen tissue. The new assays were used to characterize these enzymes in mouse liver homogenate. Because carnitine biosynthesis has been studied extensively in the rat, we compared the mouse tissue distribution of carnitine biosynthesis enzyme activities and levels of the biosynthesis metabolites with those in the rat to determine which tissues contribute to carnitine biosynthesis in these species. Surprisingly, large differences in enzyme activities were found between the rat and the mouse, whereas carnitine biosynthesis metabolite levels were very similar in both species, possibly due to the different kinetic properties of the first enzyme of carnitine biosynthesis. Also, muscle carnitine levels were found to vary considerably between these two species, suggesting that there is a metabolic dissimilarity between the mouse and the rat.