Gerard O'cuinn

Degradation of thyrotropin-releasing hormone and luteinising hormone-releasing hormone by enzymes of brain tissue.

Abstract: In this article, the enzymes of brain and associated tissues that can degrade thyrotropin‐releasing hormone (TRH) and luteinising hormone‐releasing hormone (LH‐RH) are reviewed. As both TRH and LH‐RH are considered to act as neurotransmitters or neuromodulators in the CNS, attention is paid to the subcellular location of the enzymes described and how their topographies and substrate specificities fit them to playing roles as inactivating agents for TRH and LH‐RH or as regulators of intracellular concentrations of TRH and LH‐RH. Consideration is also given to enzymes involved in biotransformation of TRH to secondary metabolites that exhibit biological activity and to enzymes involved in the metabolism of secondary metabolites.

Purification and characterization of an aminoacyl proline hydrolase from guinea-pig intestinal mucosa.

The purification of an aminoacylproline hydrolase from guinea-pig intestinal mucosa is described. The enzyme, which is an aminopeptidase has a molecular weight of 112 000 and is activated by manganese and inhibited by zinc. Unlike other aminoacylproline hydrolases this enzyme displayed a broad substrate specificity. However, it was preferentially active against dipeptides containing proline in the C-terminal position.

Active site studies on a narrow-specificity thyroliberin-hydrolysing pyroglutamate aminopeptidase purified from synaptosomal membrane of guinea-pig brain

Abstract: The effect of protein‐modifying reagents on the activity of a purified preparation of a thyroliberin‐hydrolysing pyroglutamate aminopeptidase, solubilised from synaptosomal membranes of guinea‐pig brain by treatment with papain, was investigated. The results indicated that tyrosine, histidine, arginine, and possibly lysine residues were necessary for expression of catalytic activity and that these tyrosine, histidine, and arginine residues were probably located at the active site of the enzyme. Cysteine, serine, glutamate, and aspartate residues were not involved in the expression of catalytic activity.

Immunological relationship between three intestinal peptide hydrolases and similar enzymes in various tissues of the guinea-pig and of other animals.

Previous studies from this laboratory showed that guinea-pig intestinal mucosa contains at least seven peptide hydrolases distinguishable by their substrate specificities and electrophoretic mobilities [ I] . Although these intestinal peptide hydrolases are probably responsible for the final stages of protein digestion the extent of their relationship with peptide hydrolases in other tissues of the guinea-pig is not known. Three of these peptide hydrolases, denoted (Y, /3a and y on the basis of their different electrophoretic mobilities were purified to homogeneity and characterised [2--41. These three peptide hydrolases which represent over 90% of the total dipeptide hydrolase activity of guinea-pig intestine have several features in common including overlapping substrate specificities although each peptide hydrolase specifically hydrolyses particular peptide linkages. Some relationship between the three hydrolases is suggested by their mol. wts which are 300 000, 170 000 and 112 000 for the cr, /3* and y peptide hydrolases respectively. The aim of the present study was to determine the extent to which these peptide hydrolases are immunologically related to, 1) each other, 2) enzymes with similar substrate specificities and electrophoretic mobilities in additional tissues of the guinea-pig, and 3) similar intestinal mucosal enzymes in different species. For this purpose antisera against homogeneous preparations of each of the three intestinal peptide hydrolases were used.

Purification and some structural properties of adenylate kinase from Leuconostoc mesenteroides (Lactobacteriaceae).

1. 1. Adenylate kinase was purified 234-fold from Leuconostoc mesenteroides by a procedure involving acidification, ammonium sulphate fractionation, DE 52 cellulose chromatography, Sephadex G-75 gel filtration and preparative polyacrylamide electrophoresis. 2. 2. The purified enzyme was found to be a glycoprotein of molecular weight 26,300. 3. 3. No subunits could be found and the enzyme contained only one sulphydryl group.

The degradation of peptides and aminoacyl amides by guinea-pig brain.

Abstract— The ability of guinea‐pig brain to hydrolyse peptides and aminoacyl amides was investigated. The majority of hydrolase activity against both peptides and aminoacyl amides tested was found to reside in the 30,000 g supernatant. Starch gel electrophoretograms of the 30,000 g supernatants showed that seven of the twelve peptides tested were each hydrolysed by more than one peptide hydrolase and that each of the peptide hydrolases observed were capable of hydrolysing more than one peptide. The peptide hydrolases in the 30,000 g supernatant fraction of guinea‐pig brain were found to have very similar electrophoretic mobilities to the peptide hydrolases in the 30,000 g supernatant fraction from guinea‐pig intestinal mucosa. Only one hydrolase with activity against L‐Leu‐NH, could be detected in 30,000 g supernatant of guinea‐pig brain using starch gel electrophoresis followed by staining and its electrophoretic mobility was identical to that of one of the peptide hydrolases in the same fraction.

Similarities between one of the multiple forms of peptide hydrolase purified from brush-border and cytosol fractions of guinea pig intestinal mucosa

The final stages of mammalian protein digestion are mediated by peptide hydrolases present in the mucosal cells (enterocytes) of the small intestine. These intestinal hydrolases, which occur in multiple forms with broad substrate specificity, are present in brush-border and cytosol fractions of enterocytes [l]. A topic of current interest in intestinal enzymology is whether peptide hydrolases, from brush-border and cytosol fractions of enterocytes are different. Further information on this topic, about which some confusion exists at present, is essential to provide a better understanding of protein digestion and may also help in defining the aetiology of protein malabsorption conditions such as coeliac disease (gluten-induced enteropathy) PI. Previous studies showed that many brush border and cytosol peptide hydrolases had similar electrophoretie mobilities [I]. The purpose of the present studies was to compare the properties of one of the multiple forms of peptide hydrolase recently purified and characterized from the cytosol of the enterocytes [3], with an enzyme of similar electrophoretic mobility, purified from brush-border fractions.

Localisation of a particulate luliberin hydrolysing activity in microsomal membranes of guinea pig brain.

A particulate luliberin hydrolysing enzyme has been described for guinea pig brain. Examination of subcellular fractions generated under different conditions indicated that particulate luliberin hydrolysing activity was most closely associated with the microsomal marker, rotenone-insensitive NADH cytochrome C reductase. The results obtained indicate that luliberin hydrolysing activity is not associated with synaptosomal membrane preparations and that such luliberin hydrolysing activity as is observed in synaptosomal membranes is probably the result of contamination by microsomes. The enzyme could be released from microsomes by Triton X-100 treatment and the solubilised enzyme was found to be inhibited by puromycin and sulphydryl reagents but to be unaffected by phosphoramidon, captopril, phenylmethyl sulphonyl fluoride and by chelating agents except 1,10-phenanthroline.

COMPARATIVE STUDIES OF MULTIPLE PEPTIDE HYDROLASES FROM GUINEA PIG SMALL INTESTINAL MUCOSA AND THE IMPLICATIONS OF THESE STUDIES FOR THE “MISSING PEPTIDASE” HYPOTHESIS FOR COELIAC DISEASE

ABSTRACT. Three cytoplasmic peptide hydrolases from guinea-pig small intestinal mucose, termed α,β 1 , and β 2 , have been purified and characterized. These were active against a broad and overlapping range of peptides. The β 1 and β 2 peptide hydrolases had very similar properties, although they differed in their affinities for substrates. In contrast, α distinctly differed from these in its substrate specificity, metal requirement, and other properties.