Purification and some properties of two anionic trypsins from the eel (Anguilla japonica)

Two anionic enzymes, designated as trypsins 1 and 2, were purified from the pancreas of the eel Anguilla japonica by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The final preparation of trypsin 1 was homogeneous but that of trypsin 2 still contained impurities. Both enzymes had similar pH optima of near 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. Trypsin 1 was stabilized by calcium ions but the stability of trypsin 2 was not affected by calcium ions. Both enzymes were inhibited by typical trypsin inhibitors including serine proteinase inhibitors.     

A versatile library of activity-based probes for fluorescence detection and/or affinity isolation of lipolytic enzymes

This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.     

Preparation of pancreatin and purification of lipase from hog pancreas

Pancreatin containing high activities of proteolytic enzymes, amylase and lipase was prepared from optimally autolyzed hog pancreas. About one hundred grams of pancreatin were obtained from one kilogram of hog pancreas. Lipase was purified from the pancreatin preparation through steps of mild alkaline solution extraction, removing proteolytic enzymes by affinity adsorption, first ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and secondary ammonium sulfate fractionation. By these steps, the purity of the enzyme increased 14 fold and the recovery of the enzyme activity was 33%. The purified lipase was not homogeneous and contained several contaminating proteins when examined by disc polyacrylamide gel electrophoresis.     

Inhibition of cysteine peptidase activity in ascitic fluid in pancreatic cancer patients

The work's objective is to answer the question whether there is any possibility of activity inhibition of cysteine peptidases inhibitors playing an important role in key processes accompanying cancer formation, including pancreas. There is a justified speculation that specific inhibitors of these enzymes may inhibit development of cancer processes by inhibiting their activity. In vitro studies confirmed that these enzymes in ascitic fluid were inhibited with egg whites inhibitors even to 90% of their original activity.     

Purification and properties of a hypothalamic peptide inhibiting prolactin secretion in vitro

A pure preparation of a peptide inhibiting at low (nm-pcm) concentrations a de novo synthesis of prolactin and its secretion into the medium during incubation of rat adenohypophysial tissue has been isolated from cattle hypothalamus. The biological action of the inhibitor differs from that of the already known inhibitors of adenohypophysial hormone secretion--dophamine and somatostatin. The loss of activity by the preparation after treatment with chymotrypsin is indicative of a peptide nature of the inhibitor. The amino acid composition and the N-terminal sequence of the peptide demonstrate its structural similarity to Leu-enkephaline.     

Biochemical determination of insecticides via cholinesterases. 1. Acetylcholinesterase from rat brain: functional expression using a baculovirus system, and biochemical characterization

Using a baculovirus-insect cell system, acetylcholinesterase from rat brain (rbAChE) was actively expressed and biochemically characterized and compared to the non-recombinant, tissue-derived rbAChE, establishing biochemical identity between both enzymes. Inhibition kinetics were similar for both enzymes using reversible inhibitors specific to the active and peripheral site. Inhibition rate constants of both enzymes treated with paraoxon as a reference insecticide were identical for both enzymes. As neither the catalytic parameters nor inhibition kinetics revealed any significant difference among the tissue derived and the recombinant enzyme, the baculovirus-insect expression system can be used for the preparation of recombinant rbAChE and mutants thereof.     

Localization of alkaline phosphatase and adenosine triphosphatase in the mammalian pancreas

The light microscopic histochemical localization of alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) in the mammalian pancreas is reviewed. Capillary endothelial cells usually exhibit both enzymes. ATPase is usually present in endocrine and acinar cells and absent in duct cells. APase is often found in islet cells but is almost always absent in exocrine cells. These enzymes should therefore be used with caution as markers for specific cells and organelles of the pancreas, and for monitoring diseases that might lead to the release of these enzymes from the pancreas.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Studies on lysophospholipases, V. The action of lysolecithin-hydrolyzing enzymes on lecithins and 1-acyl lysolecithins with varying fatty acid chain-length.

The activity of two purified lysolecithin-hydrolyzing enzymes on homologous series of synthetic lecithins containing two identical fatty acyl chains and of 1-acyl-lysolecithins has been measured as a function of substrate concentration. In general, enzymatic activity toward lecithins decreased with increasing chain length. Maximal hydrolysis rates for the lysolecithin series were measured with 1-dodecanoyllysolecithin. In this series increased affinities for substrates with increasing acyl-chain length was noticed. In the substrate concentration versus enzymatic velocity curves no breaks were observed at the critical micelle concentration of the various substrates. The initial site of attack during hydrolysis of short-chain lecithins was determined using 1-octanoyl-2pentanoyl-lecithin, 1-hexanoyl-2-hexyllecithin and 1 -hexyl-2-hexanoyllecithin. Both enzymes exhibited a pronounced preference for hydrolysis of the acyl ester bond at the 1-position. Especially the enzyme from beef pancreas seems to be suitable for the enzymatic preparation of 2-acyl lysolecithins from the corresponding short-chain lecithins.     

Antibody-dependent cell-mediated cytotoxicity in humans. III. Effect of protease inhibitors and substrates.

Different classes of protease inhibitors and substrates were tested for their effect on the ability of human lymphocytes to mediate antibody-dependent cytotoxicity (Ab CMC). All the inhibitors tested (serine esterase inhibitors, chloromethyl ketone derivatives of tosyl-amino acids, synthetic protease substrates), except for the naturally occurring protease inhibitors (derived from soybean, lima bean, and porcine pancreas), were able to suppress, or to reduce insignificantly, the cytotoxicity. In the absence of a direct demonstration of an esterase activity, sensitive to the action of the inhibitors in the effector lymphocytes, careful controls were used to restrict the possibility that some nonspecific effect of the drugs was being interpreted. Particularly, the dependence of the inhibition of cytotoxicity as an effect of drugs on membrane transport mechanisms or on energy metabolism was excluded. The similarity between results obtained with compounds of different chemical characteristics and different molecular mechanisms of action supports a specific effect of the inhibitor on cellular esterase(s) or possibly protease(s). The fully reversible inhibition obtained with serine esterase inhibitors suggests that the relevant enzymes are activated only after effector-target cell interaction; the irreversible effect of chloromethyl ketone derivatives, however, does not allow the participation of already activated enzymes to be excluded. The results presented in this study on the probable role of cellular esterases, on cation requirement and on the sequence of biochemical steps in Ab CMC add a new element to the analogy between this cellular phenomenon and different types of cytotoxicity or other immunologically induced cellular reactions, suggesting that the biochemical mechanisms of cytotoxicity may partly reflect a common pattern of cellular response to external stimuli.     

Microbial inhibitors of phospholipase C

Inhibitors of the Clostridium perfringens phospholipase C were prepared from the filtrates of the culture liquids of Streptomyces saracetidus and Streptomyces species using soluble and cross-linked polyelectrolytes. The technological scheme of isolation involves ultrafiltration. The inhibitors produced by the two strains had different chemical nature. The preparation obtained from Str. saraceticus was proved to be a complex of inhibitors that were separated by gel-chromatography into a major polypeptide with a molecular weight of 5500-6500 and a low-molecular weight glycopeptide. The inhibitor obtained from Str. species was found to be a high-molecular weight protein.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Venom Toxins: Plausible Evolution from Digestive Enzymes

Some hydrolytic enzymes are common to the pancreas, the mammaliansalivary glands and the snake venom glands. Phospholipase A,which is found in elapid and viperid venoms and in the mammalianpancreas, shows 29 common amino acid residues out of 118–125positions. Presynaptic neurotoxins and other venom toxins areusually composed of 2–3 units or subumts,one of whichis a phospholipase. The Vipera palaestinae two-component toxinretains its lethality when the enzyme is replaced by heterologousvenom phospholipases, but not by the pig pancreatic enzyme.This toxin is neutralized by a factor found in the blood serumof snakes, which binds to the phospholipase and inhibits itsactivity. The blood serum of snakes also neutralizes hemorrhaginsand inhibits the protease activity of the venom. It is hypothesizedthat the developing venom glands first produced enzymes thatwere already secreted by the pancreas and against which inhibitorswere present in the blood. These inhibitors facilitated theevolution of enzyme-based toxins by neutralizing any damagingsubstances that might have escaped from the venom glands.     

Characterization of monoamine oxidase isoforms in human islets of Langerhans.

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.     

Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases

Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.     

Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase.

Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.     

Identification of insulin in the human pancreatic juice

In this study, we purified insulin-like substance (ILS) in the human pancreatic juice by the combined use of affinity chromatography and radioimmunoassay (RIA). The amino acid sequence of ILS in the N-terminal region is the same as that of human insulin. The influence of the enzymes present in the pancreatic juice on the RIA procedure, was examined. Trypsin, chymotrypsin and amylase showed steep influences on radioactivity. The addition of enzyme inhibitors could not reduce pseudo-activity, but the elimination of enzymes in the pancreatic juice by ultrafiltration with the Mole-Cut (Millipore, Japan) resulted in a lowering of the pseudo-insulin activity. Affinity chromatography on Sepharose 4B coupled with anti-porcine insulin was used to capture ILS. ILS was eluted by 1 M acetic acid from the affinity column monitoring pH and the insulin activity by RIA. The amino acid sequences of two components of ILS in amino terminal region were Phe-Val and Gly-Ile-Val. This indicates that ILS obtained from human pancreatic juice was the insulin derived from endocrine secretion of pancreas.     

The immunological characterization of several human ribonucleases by using primary binding tests.

RNAases (ribonucleases), purified from four human tissues, as well as bovine pancreatic RNAase (RNAase A), were studied by immunodiffusion methods and by two different primary binding tests. The enzymes fell into two groups immunologically, those purified from plasma and pancreas in one and those from spleen and liver in the other. No antigenic cross-reaction between the two groups was detected by any of the immunoassays used. There was a slight antigenic cross-reaction between the human and bovine pancreatic RNAases. The liver and spleen RNAases were immunologically identical by all criteria used, whereas a small but consistent antigenic difference between the human plasma and human pancreas enzymes was detected. The significance of this difference between the human plasma and pancreas RNAases is discussed in relation to similarities and differences in their properties.