The major dog pancreas protein recognized by an antiserum to dog prostate kallikrein is the anionic trypsin

• 1.1. On the basis of its immunoreactivity with a polyclonal antiserum to dog prostate kallikrein in Western blot experiments, a 30 kDa protein was purified from the pancreas of the dog using ion-exchange and gel filtration chromatography.
• 2.2. That protein was identified as the anionic trypsin by its NH₂-terminal amino acid sequence.
• 3.3. The immunoreaction occurred despite an overall amino acid homology which was limited to 39% between the prostate kallikrein and anionic trypsin.
• 4.4. Otherwise, the anti-prostatic kallikrein antiserum was rather specific since it did not react with dog cationic trypsin, dog renal kallikrein and human prostate specific antigen.

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Characterization of an anionic trypsin from the eel (Anguilla japonica)

An enzyme, isolated from the pancreas of the eel Anguilla japonica and designated as anionic trypsin 1, had a molecular weight of 26,000 and an isoelectric point of 5.5. The amino acid composition of the enzyme was similar to that of bovine cationic trypsin as well as anionic trypsins from other species of fish. The enzyme was stable at pH 6 to 9 in the presence of calcium ions. Km and kcat values of the enzyme for N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester were quite similar to those of catfish anionic and bovine cationic trypsins.     

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

Comparative biochemistry of mammalian arylsulfatase C and steroid sulfatase

1. Hepatic arylsulfatase C (ASC) and steroid sulfatase (SS) from six of eleven mammals (rat, dog, baboon, cow, goat, and sheep) coeluted from DEAE-Sephacel as a single anionic species. A minor cationic peak of ASC and SS activity was also recovered from solubilized microsomes derived from the domestic cat. Characterization of the cationic activities indicated they were most likely contributed by a protein structurally related to the anionic isozyme. Properties of ASC and SS activities occurring in these seven species were most consistent with the presence of both activities in the same enzyme. 2. Guinea-pig liver SS activity was partitioned between an alkylsulfatase (hydrolyzing dehydroepiandrosterone sulfate (DHEAS)) and an arylsulfatase (hydrolyzing both estrone sulfate (E1S) and 4-methylumbelliferyl sulfate (4MUS) at a common active site). These enzymes were physically separable by ion-exchange chromatography and possessed distinct immunological and chemical properties. 3. Porcine, squirrel, and human livers possessed a major isozyme of ASC that lacked both E1S- and DHEAS-sulfatase activities. The human hepatic ASC was separable from SS by electrophoresis and was partially resolved from SS by DEAE-Sephacel chromatography. The ASC isozyme lacking SS activity was heat-labile in all three species.     

Preparation and properties of trypsin from the pyloric caeca of Pacific Ocean salmon

Trypsin from pyloric caeca of Pacific salmon was purified by affinity chromatography of the water extract on hexamethylenediamine-glycidylmethacrylate-cellulose. A protein band with a molecular weight of 22.5 kDa was found on SDS-electrophoresis in PAG. The protein band was homogeneous according to isoelectrofocusing in PAG (pI 4.0). The amino acid composition of the enzyme is typical of trypsin anionic forms; the major difference from the cationic forms is the lower content of lysine. The differences in properties caused by change of the enzyme molecule charge are similar to those observed in cationic trypsin when the lysine epsilon-amino groups of the latter are modified (change of pI, shift of the pH-optimum towards basic values, increase of stability to autolysis). Some natural trypsin inhibitors of the different origin suppressed the enzyme activity of trypsin from Pacific salmon in typical stoichiometric ratios. An unusual interaction of the enzyme with the specific inhibitor N-L-tosyl-L-lysine chloromethyl ketone was observed.     

Immunoreactive trypsins in sera from dogs before and after induction of experimental pancreatitis

Radioimmunoassays for anionic and cationic dog trypsins are described. Characterization of the immunoreactivities in sera from fasting dogs demonstrated the presence of the two proenzymes only. Fasting sera from 10 dogs contained anionic and cationic trypsinogen in concentrations between 17-110 micrograms/l and 7-19 micrograms/l, respectively. Induction of experimental pancreatitis in dogs was accompanied by a large increase of immunoreactive anionic and cationic trypsins in the circulation. During the progress of the pancreatitis, immunoreactive trypsin with larger molecular weight than trypsinogen appeared. This high molecular weight immunoreactive trypsin was not seen in serum after intravenous injection of pancreatic juice in dogs. The high molecular weight immunoreactive trypsin probably consists of trypsin in complex with protease inhibitors. In vitro studies showed that the immunoreactivity of trypsin decreased considerably after binding to alpha 1-antitrypsin or alpha-macroglobulins.     

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes

Temperature dependences of kinetic constants (k _cat and K _m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of coldadapted fishes. The anionic and cationic trypsins were found to differ in organization of the S₁ -substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S₁) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K _m value) to the anionic and cationic trypsins and on the catalytic activity k _cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.     

Detection of prophospholipase A2 in human spermatozoa

Prophospholipase A2 (proPA2) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C4) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A2 (PA2) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C4 column. The proenzyme was separated from active PA2 on the C4 column. Human sperm proPA2 exhibited a less cationic charge than active PA2 on the SP 5PW column. Porcine pancreatic proPA2 had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA2. The purification procedure resulted in the isolation of proPA2 which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA2 of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA2 exists in human spermatozoa.     

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.     

Design of novel cationic ligands for the purification of trypsin-like proteases by affinity chromatography

A number on new cationic ligands have been designed and synthesized for the selective resolution an purification of the trypszin-like proteases. A series of ligands based on 4-[2′-methyl-4′-(2″,4″-dichloro-1″,3″,5″-triazin-6-ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4-aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution ad following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.     

Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotropic virus infectivity and increased enzyme level after virus infection.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. In search of such target processing proteases in the airway, we recently found a new candidate trypsin-like processing protease in rat lungs, which was induced by Sendai virus infection, and identified as ectopic rat anionic trypsin I. On SDS/PAGE under reducing and nonreducing conditions, the purified enzyme gave protein bands corresponding to 29 and 22 kDa, respectively, i.e. at the same positions as rat pancreatic anionic trypsin I. It exhibited an apparent molecular mass of 31 kDa on molecular sieve chromatography and its isoelectric point was pH 4.7. The amino-acid sequences of the N-terminus and proteolytic digest peptides of the purified enzyme were consistent with those of rat pancreatic anionic trypsin I. Its substrate specificities and inhibitor sensitivities were the same as those of the pancreatic enzyme. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and hemagglutinin of human influenza A virus, and potentiated the infectivity of Sendai virus in the same dose-dependent manner as the pancreatic one. Immunohistochemical studies revealed that this protease is located in the stromal cells in peri-bronchiolar regions. These results suggest that ectopic anionic trypsin I in rat lungs induced by virus infection may trigger virus spread in rat lungs.     

Comparative biochemistry of mammalian arylsulfatases A and B

1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, Mr = 67 +/- 2 kDa), an apparent aggregate (dog, whale, pig; Mr = 192 +/- 10 kDa), or both (rat, mouse; monomeric Mr = 57 +/- 2 kDa; apparent dimeric Mr = 114 +/- 3 kDa). 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. Opossum arylsulfatases A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations.     

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin,reveals its resistance to proteinaceous trypsin inhibitors

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.     

Isolation and characterization of a cDNA encoding rat cationic trypsinogen

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.     

The Preparation of Trypsins and Chymotrypsins From Bovine and Porcine Residues After Insulin Extraction

Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe₈₉-Lys₉₀.     

Enzymatic characterization of arginine esterase from dog seminal plasma

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Binding of Fab-horseradish peroxidase conjugates by charge and not by immunospecificity

The influence of horseradish peroxidase (HRP) charge on Fab-HRP conjugates was investigated. Rabbit nonimmune Fab coupled via periodate or glutaraldehyde to Sigma HRP type VI (pI greater than 10) were cationic (positively charged) as determined by analytical isoelectric focusing. These conjugates and HRP type VI alone stippled the basal laminae and collagen fibers in Bruch's membrane of the rat eye in a pattern identical to anionic (negative) sites. Binding was not present after the anionic sites were removed by enzyme digestion prior to immunolabeling or when HRP type VIII (anionic with pI 3.6) was used in an Fab-HRP conjugate or in an unbound form. These results indicate that anionic HRPs should be used in Fab-HRP preparations if a nonspecific binding to anionic sites is possible.     

Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms.

Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl substrate were attacked most rapidly and their hydrolyses were stimulated by Ca2+. The 2-naphthylamide derivatives of neutral and basic amino acids were also hydrolysed by aminopeptidase A, but at rates about two orders of magnitude lower, and Ca2+ was inhibitory. The possibility that these atypical substrates were hydrolysed by traces of aminopeptidase M (EC 3.4.11.2) contaminating the preparation could be excluded on several grounds. Aminopeptidase A was sensitive to inhibition by chelating agents and the inactive enzyme could be reactivated by Ca2+ or Mn2+. Atomic absorption spectrophotometry revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar protein.     

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.