Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.     

Trypsin inhibitors in guinea pig plasma: isolation and characterization of contrapsin and two isoforms of alpha-1-antiproteinase and acute phase response of four major trypsin inhibitors

Contrapsin and two isoforms, F (fast) and S (slow), of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of alpha-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of alpha-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and alpha-macroglobulin showed moderate (1.5 to 2.3-fold) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different alpha-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of alpha-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i.e., contrapsin, alpha-1-antiproteinase, alpha-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.     

Synthesis of contrapsin and alpha-1-antiproteinase in inflamed and tumor-bearing mice

In mice bearing ascites tumors, such as Ehrlich, L1210, Meth-A, and P-815, the plasma levels of contrapsin and alpha-1-antiproteinase remained virtually unchanged. However, the total body pools of these proteins as well as their hepatic mRNA levels increased severalfold, and isoelectrofocusing patterns of these proteins shifted to the lower pH side. Under the same conditions, plasma albumin level decreased by 25%, but its total body pool and hepatic mRNA level increased severalfold. On the other hand, induction of acute phase reaction by injection of bacterial lipopolysaccharide caused differential effects on the two proteinase inhibitors: hepatic translatable mRNA for contrapsin was doubled while that for alpha-1-antiproteinase remained unchanged.     

Serum trypsin inhibitory activity and alpha-1-antitrypsin level in liver diseases

Alpha-1-antitrypsin level and serum trypsin inhibitory activity were measured in patients with viral hepatitis, chronic hepatitis and liver cirrhosis. The most pronounced discrepancy between these two parameters were observed in patients with liver cirrhosis: the increase of alpha-1-At level was not accompanied by adequate increase of trypsin inhibitory activity. Some mechanisms potentially responsible for this discrepancy are discussed.     

Purification, characterization, and acute phase response of plasma alpha-1-antiproteinase in the hamster, Mesacricetus auratus.

1. alpha-1-Antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) with a molecular mass of 60 kDa was purified to apparent homogeneity from hamster plasma. 2. It inhibited elastase, chymotrypsin and trypsin, but did not significantly affect pancreatic kallikrein, plasma kallikrein or plasmin. 3. It has the same N-terminal heptapeptide sequence as that of rat alpha-1-antiproteinase. 4. Its plasma level decreased after injection of bacterial lipopolysaccharide.     

The molecular stoichiometry of trypsin inhibition by human alpha-1-proteinase inhibitor

The stoichiometry of interaction of human alpha-1-proteinase inhibitor with porcine trypsin has been determined using a highly purified preparation of inhibitor. In contrast to the reports of others, one mole of alpha-1-proteinase inhibitor was found to inhibit two moles of trypsin. Disc gel electrophoresis indicates that the 2:1 complex is preferentially formed even when free alpha-1-proteinase inhibitor is still present.     

Human alpha-1-microglobulin is covalently bound to kynurenine-derived chromophores

Alpha-1-microglobulin carries a set of covalently linked chromophores that give it a peculiar yellow-brown color, fluorescence properties, and both charge and size heterogeneity. In this report it is shown that these features are due to the adducts with the tryptophan metabolite, 3-hydroxykynurenine, and its autoxidation products and that the modification is more pronounced in the protein isolated from urine of hemodialyzed patients. The light yellow amniotic fluid alpha-1-microglobulin acquires the optical properties and charge heterogeneity of the urinary counterpart following incubation with kynurenines. The colored amino acid adducts of urinary and amniotic fluid alpha-1-microglobulins were separated by chromatography after acid hydrolysis and analyzed by mass spectrometry. Human serum albumin samples, native and treated with 3-hydroxykynurenine in the presence of oxygen, were used as a control. The retention times and mass fragmentation products were compared, and a lysyl adduct with hydroxantommathin was identified in the urinary alpha-1-microglobulin and in the modified albumin samples. The more extensive modification of the urinary protein appears to be correlated with uremia, a condition in which the catabolism of tryptophan via the kynurenine pathway is increased, and the consequent rise in the concentration of its derivatives is accompanied by the oxidative processes due to the hemodialysis treatment. The oxidative derivatives of 3-hydroxykynurenine, which are known to act as protein cross-linking agents, are the likely cause of the propensity of urinary alpha-1-microglobulin to form dimers and oligomers. This process, as well as the redox properties of these metabolites, may contribute to the toxic effects of the kynurenine species.     

Cloning and sequencing of cDNA coding for rabbit alpha-1-antiproteinase F: amino acid sequence comparison of alpha-1-antiproteinases of six mammals

Rabbit liver cDNA coding for alpha-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit alpha-1-antiproteinase F showed 74 and 64% homology to human alpha-1-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of alpha-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.     

Radioimmunoassay of human plasma trypsin.

A radioimmunoassay has been developed for the determination of human trypsin (3.4.21.4) in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard and for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity.     

Choline acetyltransferase-like activity bound to neuronal plasma membranes

A form of CAT-like activity was found bound present in rat brain synaptosomal membranes which could be recovered in the Triton X-114 phase. The enzyme activity was slightly activated by NaCl, had a pH maximum around 8 and showed a temperature dependence with a Q₁₀ of 2.28. It was inhibited 100% by 10^–6 M naphthyl vinyl pyridinium but not by 10^–5 M diisopropyl phosphofluoridate. The kinetics of this bound form of CAT were similar to the soluble form of the enzyme. TheK _m was 405±58 M for choline and 62±8 M for AcCoA. Five isoelectric forms were found with pH's of 4.55, 6.05, 7.06, 7.36, and 8.00 which is in contrast to the three isoelectric forms found of the soluble enzyme in rat brain. The presence of a CAT-like activity in the plasma membrane was confirmed with experiments performed using intact synaptosomes and intact cells in culture. Acetylcholine, synthesized from radioactive AcCoA by intact rat brain synaptosomes, was recovered in the incubation medium and only in the presence of exogenous choline or when the production of choline was stimulated by oleate via the activation of phospholipase D. This was also seen in experiments with intact pheochromocytoma cell cultures (PC 12) which synthesize acetylcholine that was recoverved in the incubation medium. Acetylcholine formation in the presence of choline and AcCoA was stimulated in cells that had been grown in the presence of nerve growth factor (NGF). The localization of 1% of CAT activity in a transbilayer position in the plasma membrane, could suggest a possible role of this enzymatic form in the regulation of acetylcholine synthesis.     

Nucleophilic cleavage of the complex between human alpha-1-antitrypsin and porcine pancreatic elastase

Several nucleophilic amines were examined to determine their ability to split the alpha-1-antitrypsin-elastase complex. Hydrazine and hydroxylamine, their methyl derivatives, and methylamine were tested in the pH range of 8 to 10.6. Only hydrazine and methylamine produced complete and clean cleavage of the complex into its inhibitor and enzyme components. When [¹⁴C]-methylamine at pH 10.6 was used about 0.3 mol of the nucleophile was specifically bound per mol of the inhibitor component. An interfering reaction between methylamine and the native inhibitor was controlled by preincubation with unlabeled methylamine.     

Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-specific antigen), at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high Mr basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K(m) values were in the 10(2)-10(3) m(-1) x s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex vivo. PAP cleavage sites differed from those of hK3 and were mainly at P1 = Gln residues or between residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP. Purified human semenogelin was cleaved by purified and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identified ex vivo by analysis of seminal plasma.     

Antigen-binding activity,structural characteristics and use of monoclonal antibodies to human alpha-1-microglobulin

Four monoclonal antibodies (mAbs), G6, F9, H8, and B2, against human alpha-1-microglobulin (A1M) have been produced and characterized. The parameters of affinity (K_p ～ 10⁹ M^?1), epitope specificity (the additively binding G6/F9, G6/H8, G6/B2, F9/H8, and F9/B2 pairs), and the observed effect of reversibility of structural changes induced by chemical agents allow use of these mAbs in biospecific methods of A1M purification and quantitative determination. The application of mAbs to an A1M enzyme immunoassay (analytical sensitivity—0.5 μg/l) and one step isolation of pure A1M by immunoaffinity chromatography was described.