Pig Enzyme Research Articles

Guinea pig 11β-hydroxysteroid dehydrogenase type 1: primary structure and catalytic properties☆

The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme is responsible for the interconversion of glucocorticoids and their inactive metabolites, and thus modulates the intracellular level of bioactive glucocorticoids. The present study was designed to clone and characterize 11β-HSD1 in the guinea pig, a laboratory animal known for resistance to glucocorticoids. The cDNA encoding guinea pig 11β-HSD1 was cloned by a modified 3′-RACE (rapid amplification of cDNA ends) protocol using the hepatic RNA as template. The cloned cDNA encodes a protein of 300 amino acids that shares 71 to 74% sequence identity with other known mammalian 11β-HSD1 proteins. Sequence comparison analysis revealed that the deduced guinea pig 11β-HSD1 was longer, by eight amino acids at the C terminus, than those of other mammals. Moreover, one of the two absolutely conserved consensus sites for N-glycosylation was absent. To examine the functional significance of these structural changes, we also characterized 11β-HSD1 activity in the hepatic microsomes. Although the guinea pig hepatic enzyme was NADP(H)-dependent and reversible, it displayed equal affinity for cortisol and cortisone (apparent K m for both substrates was 3 μM). This is in marked contrast to 11β-HSD1 in other mammals whose affinity for cortisone is approximately 10 times higher than that for cortisol (apparent K m of 0.3 vs. 3.0 μM). The apparent lower affinity of the guinea pig enzyme for cortisone would suggest that the intracellular bioformation of cortisol from circulating cortisone may be less efficient in this species. Northern blot analysis and RT-PCR revealed that the mRNA for 11β-HSD1 was widely expressed in the adult guinea pig but at low amounts. In conclusion, the present study has identified distinct features in the deduced primary structure and catalytic function of 11β-HSD1 in the guinea pig. Thus, the guinea pig provides a useful model in which the structural determinants of catalytic function of 11β-HSD1 may be studied.

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation.

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc(3)-Man(9)-GlcNAc(2) from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of approximately 48.9 kDa (OST48), approximately 68.7 kDa (ribophorin I) and approximately 69.3kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein. Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible approximately 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the -3/-5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the -3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.

Selection of cleavage site by mammalian tRNA 3′ processing endoribonuclease

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) removes 3′ trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3′ tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNA Arg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3′ trailer are sufficient for recognition by 3′ tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp ( N is less than 12) were cleaved 12— N and 13— N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3′ tRNase.

The influence of supplemental alpha‐galactosidase and phytase in a vegetable ration for dogs on the digestibility of organic components and phytate phosphorus

The use of dry feeds for dogs is increasing for different reasons: in comparison to wet canned feed it is easier to handle, the amount to be given is smaller and the daily cost is less. In producing a dry pellet more vegetable ingredients are used that can contain several antinutritional factors. Some of them can be eliminated by heating. Others like galactosides and phytates are heat‐resistant but can be broken down by adding the appropriate enzymes. The specific enzymes are indeed not secreted by the animal itself. In farm animals, e.g. pigs, alpha‐galactosidase has been introduced in order to enhance the energetic value of these alphagalactosides (Veldman et al. 1993). In companion animals this enzyme is more often indicated as an antiflatulence agent, because the main alpha‐galactosides present in legume seeds (stachyose, raffinose) are known as flatulence components (Fleming 1981).These oligosaccharides do not necessarily imply an overall lower nutrient digestibility. Zuo et al. (1996) did not prove a significant difference in digestibility between conventional and low oligosaccharide soybean meal in dog rations. Others however, (Wiernusz et al. 1995) reported the highest digestibilities and the best stool quality of those rations that contained the least amount of soybean oligosaccharides.On the other hand, phytates (phospho‐inositols) are known as complexing agents of several divalent cations (Fe, Mg, Zn, Ca) and are themselves considered as a phosphorus source, unavailable to monogastric animals. Phytase is the appropriate enzyme, which is not secreted by the animal itself, but, when added to the diet, can enhance the bio‐availability of several minerals and trace elements (Rimbach et al. 1994) by hydrolysing the phospho‐inositol molecule.The aim of the experiment, reported here, is to investigate the influence of the addition of alpha‐galactosidase, phytase, or both to a nearly complete vegetable ration, consisting of a relatively high content of galactosides and phytates. The combination of both enzymes was carried out because Wenk et al. (1993) reported a possible interaction between a carbohydrase and a phytase enzyme in pigs.

Interaction of Pig Kidney and Lentil Seedling Copper-Containing Amine Oxidases with Guanidinium Compounds

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (K(i) =1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a K(i) value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human coeliac disease.

Coeliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+ BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.

Effect of dietary supplementation of raw and extruded grass pea seeds (<i>Lathyrus sativus</i> L.) on activity of pancreatic enzymes in pigs

1. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed C.D Hanbury, C.L White, B.P Mullan, K.H.M Siddique Animal Feed Science and Technology CrossRef

Cardiac xenotransplantation

The severe shortage of human donor hearts has prompted several investigators to develop alternative strategies using cross-specie organs or xenografts. Unlike human allotransplantation, in which the important antigenic differences between donor and recipient are confined to the major histocompatibility and blood group antigens, xenotransplantation is confronted with the potential for multiple antigenic differences. In the pig-to-primate model of xenotransplantation, the primary obstacle to cross-species transplantation has been hyperacute rejection mediated by complement fixing antibodies directed against galactose α1,3-galactose (Gal α1,3-Gal) epitopes on the pig endothelium. Conventional immunosuppression is unable to overcome hyperacute rejection; however, recent efforts in molecular biology have focused on genetically engineering porcine donors to express human proteins in their tissue. Transgenic pigs that express human complement regulatory proteins on their endothelium have been developed. Heterotopic transplantation of these transgenic donor hearts have had only moderate success. Alternative approaches attempt to eliminate the Gal α 1,3-Gal epitopes by genetically “knocking out” the enzyme necessary for its synthesis, or to reduce the expression of Gal α 1,3-Gal epitopes by genetically inserting enzymes that redirect precursor molecules into alternative synthetic pathways. The technology to knock out the necessary enzymes in pigs is not yet available; however, pigs expressing the H-transferase gene have been developed and show reduced levels of Gal α1,3-Gal epitopes. Although this “new breed” of transgenic pigs may overcome the barrier of hyperacute rejection, special strategies will need to be developed that target the next barrier of xenograft rejection.

Recombinant Human and Mouse Purple Acid Phosphatases: Expression and Characterization

The mammalian purple acid phosphatases (also called tartrate-resistant acid phosphatases) are expressed primarily in actively resorbing osteoclasts and activated macrophages. The enzymes are characterized by the presence of a binuclear iron center at the active site. Recent studies on transgenic mice lacking purple acid phosphatase implicate the osteoclast enzyme in both bone resorption and bone mineralization. To characterize the mammalian enzymes in more detail, particularly with respect to their substrate specificity at the low pH of the osteoclastic resorptive space (2.5–3), we have purified the recombinant human and mouse enzymes from baculovirus-infected insect cells. The properties of the recombinant mouse enzyme are compared with those of the nonrecombinant enzyme isolated from mouse spleen. The kinetics of hydrolysis of the substratesp-nitrophenyl phosphate, phosphotyrosine, and pyrophosphate and a phosphotyrosyl peptide by the recombinant human and mouse enzymes and the nonrecombinant mouse and pig enzymes were analyzed. For all the enzymes the ratiokcat/Kmwas typically ∼106m−1s−1and was higher at pH 2.5 than at 4.9. The increase was attributable to a large decrease inKmat the lower pH value. The results indicate that the enzyme exhibits high catalytic efficiency toward substrates such as pyrophosphate and acidic phosphotyrosine-containing peptides, particularly at low pH values typical of the bone resorptive space. The implications of the results for the physiological function of the enzyme are discussed.

The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,.

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.

Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a peroxisomal targeting signal 2

Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes. The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98%. The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme. Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information. This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase. One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence. An unexpected homology with d-lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed. © 1997 Elsevier Science B.V. All rights reserved.

Cardiac xenotransplantation.

The severe shortage of human donor hearts has prompted several investigators to develop alternative strategies using cross-specie organs or xenografts. Unlike human allotransplantation, in which the important antigenic differences between donor and recipient are confined to the major histocompatibility and blood group antigens, xenotransplantation is confronted with the potential for multiple antigenic differences. In the pig-to-primate model of xenotransplantation, the primary obstacle to cross-species transplantation has been hyperacute rejection mediated by complement fixing antibodies directed against galactose alpha 1,3-galactose (Gal alpha 1,3-Gal) epitopes on the pig endothelium. Conventional immunosuppression is unable to overcome hyperacute rejection; however, recent efforts in molecular biology have focused on genetically engineering porcine donors to express human proteins in their tissue. Transgenic pigs that express human complement regulatory proteins on their endothelium have been developed. Heterotopic transplantation of these transgenic donor hearts have had only moderate success. Alternative approaches attempt to eliminate the Gal alpha 1,3-Gal epitopes by genetically "knocking out" the enzyme necessary for its synthesis, or to reduce the expression of Gal alpha 1,3-Gal epitopes by genetically inserting enzymes that redirect precursor molecules into alternative synthetic pathways. The technology to knock out the necessary enzymes in pigs is not yet available; however, pigs expressing the H-transferase gene have been developed and show reduced levels of Gal alpha 1,3-Gal epitopes. Although this "new breed" of transgenic pigs may overcome the barrier of hyperacute rejection, special strategies will need to be developed that target the next barrier of xenograft rejection.

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases

Diadenosine 5′,5‴- P 1, P 4-tetraphosphate (Ap 4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5′,5‴- P 1, P 4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap 4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap 4A hydrolase ( K m = 0.8 μM), being sensitive to inhibition by fluoride ( K i = 24 μM) and adenosine 5′-tetraphosphate ( K i = 10 nM) and yielding ATP and AMP as products. A low K m Ap 4A hydrolase ( K m = 0.3 μM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of K m for Ap 4A and K i for adenosine 5′-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequence comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.

Development of digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and pancreas.

The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

Influence of Inorganic and Organic Dietary Zinc on Its Concentration in Blood Serum, Bones and Hair, and Catalytical Activity of Selected Serum Enzymes in Pigs

Rupic. V .. L. Ivandija. S. Luterotti. M. Dominis-Kramaric: lnfiuence of Inorganic and Organic Dietary Zinc on its Concentration in Blood Serum, Bones and Hair, and on Catalytical Activity of SOllie Sen/III En~Yllles in Pigs. Acta vet. Bmo 1997.66: 75-85. The influence of dietary zinc originating from either inorganic or organic source on zincdepleted pigs was investigated. Within the 30 d period preceding the trial. all the animals (German Landrace x Pietrain x Large White x Swedish Landrace weaned crossbreds) were depleted of zinc and divided into three groups. 14 animals in each. During the experimental fattening period (105 d). the control group received no extra zinc. whereas the two trial groups were fed a diet with zinc supplement either as inorganic salt (ZnS04) or metalo-organic chelate (zinc methionate). On average. the respective supplements contained 84.3 mg Zn.kg· l diet and 40.9 mg Zn.kgl diet. Evaluation of the nutritional effects of zinc was based on its concentration in the blood serum, bone and hair. Moreover. the effect of zinc on catalytical activity of alkaline phosphatase. aspartate aminotransferase. alanine aminotransferase and gamma-glutamyltransferase in serum was studied. A decrease of catalytical activity of both aspartate aminotransferase and alanine aminotransferase was found along with signs of parakeratosis on the skin of control animals but no correlation with serum zinc was established. In contrast to this finding. a linear correlation between catalytical activity of alkaline phosphatase and of gamma-glutamyltransferase and serum zinc level was found with the respective correlation coefficients of 0.90 and -0.72. Nutritional efficiency of diet zinc was evidenced by both higher (P < 0.05) zinc level in blood serum. bone and hair and by the state of hepatobiliary tract and bones. and favourable performance results of trial animals compared to controls. None of trial animals suffered from any health disturbances. Progressive parakeratosis appeared. however. in control animals and lasted during the second month of the trial. It was accompanied by deterioration of performance data of the affected animals. Our data suggest that bioavailability of zinc from the chelate was better than of that derived from inorganic salt. particularly in the period of rapid growth. This presumbaly resulted in reduced elimination of zinc. Based on better nutritional efficiency in pigs and on ecological benefits. zinc methionate should be preferred to ZnS04 as a zinc containing diet additive. Growth, pigs. ~illc sulphate. ~illc methionate. bioavailability, nutrition The improvement of body mass gain and carcass quality are among major goals of all pork producers. One of the ways to achieve this is to provide the animals with necessary minerals from the sources of favourable bioavailability. Circulation of zinc and its biological importance in animals (cellular replication. lung tissue CO, release, sexual maturation, wound healing. fertility and reproduction) have been extensively investigated. Zinc is an essential trace element, and it is an integral part of some 300 enzymes (carbonic anhydrase, alkaline phosphatase, alcohol dehydrogenase, RNA and DNA polymerases, 5-nucleotidase, etc.) which take part in protein synthesis, metabolism of carbohydrates and nucleic acids (Mi I ne 1994). It is assumed that Zn derived from compounds with amino acids stimulates

Functional expression of the guinea pig 11b-hydroxylase in COS-1 cells.

We expressed a guinea pig 11 beta-hydroxylase cDNA (1) in COS-1 cells. In order to find the optimal expression system we compared three expression plasmids, each driven by a different promoter. Although promoters exhibited different transcriptional activities this did not result in different enzymatic activities. Upon cotransfection with bovine adrenodoxin a 5-fold increase of enzyme activity was achieved. A comparison with the bovine 11 beta-hydroxylase clearly demonstrated that the guinea pig enzyme was not able to produce significant amounts of 18-hydroxylated and 18-oxidized products from deoxycorticosterone under the experimental conditions used.

Guinea pig lung tryptase: Localisation to mast cells and characterisation of the partially purified enzyme

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to α1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 ± 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 ( N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride), but not by α1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel of 10 tryptic p-nitroanilide peptide substrates. Kinetic constants were determined for two of these: with l-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar K m but a 5-fold lower k cat than human tryptase, and with l-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower K m and a 30% greater k cat than its human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the K m towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions of tryptase in vivo, provided cognizance is taken of the differences that do exist.

Purification and characterization of human endopeptidase 3.4.24.16. Comparison with the porcine counterpart indicates a unique cleavage site on neurotensin

We have purified and characterized human brain endopeptidase 3.4.24.16. The enzyme behaved as a 72 kDa protein and belonged to the metalloprotease family. Human endopeptidase 3.4.24.16 cleaved neurotensin at a unique site at the Pro 10-Tyr 11 bond, leading to the formation of neurotensin(1–10) and neurotensin(11–13). The kinetic parameters displayed by human endopeptidase 3.4.24.16 towards a series of natural neuropeptides indicated that bradykinin was the most efficiently proteolysed. Angiotensin I, dynorphins 1–8 and 1–9 and substance P also behaved as good substrates while neuromedin N, angiotensin II, leucine and methionine enkephalin and neurokinin A resisted degradation by human endopeptidase 3.4.24.16. We have purified the porcine counterpart of endopeptidase 3.4.24.16 and compared its ability to cleave neurotensin with that of the enzyme from human origin. It appeared that, besides a major production of neurotensin(1–10), an additional formation of neurotensin(1–8) was observed with the pig enzyme, suggesting a cleavage of neurotensin not only at the Pro 10-Tyr 11 bond but also at the Arg 8-Arg 9 peptidyl bond. The latter cleavage appeared reminiscent of endopeptidase 3.4.24.15 since this peptidase was reported to cleave neurotensin at the Arg 8-Arg 9 bond. Our study indicated that neurotensin(1–10) formation by porcine endopeptidase 3.4.24.16 could be potently blocked with the selective endopeptidase 3.4.24.16 dipeptide inhibitor Pro-Ile without interfering with neurotensin(1–8) formation. By contrast, the formation of the latter product was highly potentiated by dithiothreitol and inhibited by the endopeptidase 3.4.24.15 inhibitor Cpp-Ala-Ala-Tyr-pAB, two effects that were not observed for neurotensin(1–10) production. Altogether, our results indicate that porcine endopeptidase 3.4.24.16 cleaves neurotensin at a unique site, leading to the formation of rteurotensin(1–10) and that the production of neurotensin(1–8) is due to contaminating endopeptidase 3.4.24.15.

Crystal structures of the wild type and the Glu376Gly/Thr255Glu mutant of human medium-chain acyl-CoA dehydrogenase: influence of the location of the catalytic base on substrate specificity.

Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 A resolution, respectively. The catalytic base responsible for the alpha-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its omega-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C alpha atom of the substrate and the distance between the Glu255 carboxylate and the C alpha atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C alpha-C beta bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.

Modulation of the activity of human 17 alpha-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.

Using NADPH-cytochrome P-450 reductase as electron donor the homogeneous pig 17 alpha-hydroxylase-17,20-lyase (CYP17) was shown to catalyse the conversion of delta 5, as well as delta 4, steroids (pregnenolone and progesterone respectively) predominantly into the corresponding 17 alpha-hydroxylated products. The latter were then cleaved by the lyase (desmolase) activity of the enzyme into androgens. Cytochrome b5 stimulated both these activities, but its most noticeable effect was on the formation of delta 16-steroids, which compulsorily required the presence of cytochrome b5. These results on the pig enzyme confirm the original findings [Nakajin, Takahashi, Shinoda and Hall (1985) Biochem. Biophys. Res. Commun. 132, 708-713]. The human CYP17 expressed in Escherichia coli [Imai, Globerman, Gertner, Kagawa and Waterman (1993) J. Biol. Chem. 268, 19681-19689] was also purified to homogeneity and was found to catalyse the hydroxylation of pregnenolone and progesterone without requiring cytochrome b5. Like the pig CYP17, the human CYP17 also catalysed the cytochrome b5-dependent direct cleavage of pregnenolone into the delta 5,16-steroid, but unlike it the human enzyme did not cleave progesterone at all. 17 alpha-Hydroxypregnenolone was, however, cleaved into the corresponding androgen but only in the presence of cytochrome b5. 17 alpha-Hydroxyprogesterone was a poor substrate for the human CYP17; although it was converted into androstenedione in the presence of cytochrome b5 its K(m) was 5 times higher and Vmax. 2.6 times lower than those for the hydroxylation of progesterone. The endocrinological and mechanistic implications of these results are discussed.