Corey Largman

Proteolysis of parathyroid hormone in vitro by sera from acute pancreatitis patients.

Abstract The degradation of 125I-labeled parathyroid hormone in vitro has been investigated in order to test the hypothesis that proteolytic degradation of PTH may be related to the hypocalcemia associated with acute pancreatitis. A polyacrylamide gel electrophoresis technique has been employed to separate bovine PTH (1-84) from lower molecular weight peptides produced by proteolytic cleavage. It has been demonstrated that trypsin bound to the plasma protease inhibitor α2-macroglobulin rapidly degrades PTH and destroys its biological activity in vitro in a kidney cortex adenyl cyclase assay system. Substantial PTH degrading activity was also detected in serum samples obtained from nine patients with severe acute pancreatitis. In contrast, significant PTH degradation could not be detected in normal serum or plasma. Furthermore, no degradation was observed with sera from two patients with mild pancreatitis due to biliary tract stones or four patients with hyperamylasemia but no evidence of pancreatic disease. The PTH degrading activity in patient sera could be blocked by the combination of two peptide chloromethylketones that specifically inhibit the pancreatic endopeptidases trypsin, chymotrypsin, and elastase 2. These results suggest that the FTH degradation assay represents a sensitive test for biologically relevant proteolytic activity in acute pancreatitis, and that it can specifically detect molecular forms of the pancreatic proteases in blood reflecting the intrapancreatic activation of zymogens in the course of pancreatic inflammation.

Human carboxypeptidase B. II. Purification of the enzyme from pancreatic tissue and comparison with the enzymes present in pancreatic secretion

Carboxypeptidase B (peptidyl-L-lysine (-L-arginine) hydrolase, EC 3.4.12.3) has been isolated and purified to apparent homogeneity from activated extracts of human pancreas tissue. The purified enzyme has been shown to be a single polypeptide of 34 000 daltons. In this respect the enzyme from pancreatic tissue, designated native human carboxypeptidase B, differs from the two forms present in human pancreatic juice (fractions I and II), both of which are composed of two polypeptides of approximately 24 000 and 9000 daltons. In addition, the three forms of human carboxypeptidase B differ in electrophoretic mobility in polyacrylamide gel electrophoresis and in chromatographic behavior on DEAE-cellulose. Two immunological methods, micro-complement fixation and radioimmunoassay, have shown a high degree of structural similarity between the three forms of human carboxypeptidase B. Micro-complement fixation experiments indicate that the amino acid sequences of the three enzymes differ by less than one percent. In vitro digestion studies have indicated that trypsin alone is sufficient to convert native carboxypeptidase B to carboxypeptidase B II. However, no combination of trypsin, chymotrypsin, and/or elastase was capable of converting native carboxypeptidase B to carboxypeptidase B I in vitro.

Effects of Cholecystokinin, Food Intake and Cephalic Stimuli on Plasma Levels of Amylase, Lipase, and Immunoreactive Cationic Trypsinogen in Rats

Plasma levels of amylase, lipase, and immunoreactive cationic trypsinogen (ICT) were monitored in conscious rats to study the effects of cholecystokinin octapeptide (CCK-8) plus secretin administration, cephalic stimuli, and food intake. Stepwise increasing doses of CCK-8 (1, 5, 15, 30 Ivy dog units: IDU/kg/h) caused significant dose-related increases in plasma levels of each enzyme in a similar manner as those previously observed for response patterns of CCK-induced exocrine protein secretion in the same species. ICT showed the greatest response to CCK-8 with a maximal concentration 30 times above basal levels resulting in the steepest slope of the dose-response curve. Plasma lipase showed a maximal response that was 5 times above the basal level, while the plasma amylase level was increased only by 50% at the maximal response. Computed ED50 of CCK-8 for each enzyme confirmed this relative sensitivity of the response: 3.0, 8.7, 11.0 IDU/kg/h for ICT, lipase, and amylase, respectively. Plasma levels of amylase and lipase did not change significantly in response to the intake of either a liquid diet or fiber pellets containing no caloric value. Plasma ICT levels, however, were elevated significantly by 23 and 53% at the time when the liquid diet or fiber pellets were given and when cephalic stimulation appeared maximally induced. The increased levels declined thereafter and were no longer significantly different from the basal levels for postprandial 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities of circulating immunoreactive pancreatic anionic trypsinogen in cystic fibrosis: An assay artifact due to cross-reacting serum antibodies

In patients with CF, serum pancreatic cationic trypsinogen has proven to be useful for newborn diagnostic screening and also as a test of pancreatic function in the older patient. However, an assay for serum anionic trypsinogen is of no value as a test of pancreatic function in CF due to an apparent artifactual elevation of this enzyme in some patients. In this study, we evaluated the extent of the abnormality in the anionic trypsinogen assay and also elucidated the nature of the interfering material. CF patients were grouped according to the presence (pancreatic insufficiency) or absence (pancreatic sufficiency) of steatorrhea. In CF infants, both serum cationic and anionic trypsinogen levels were greatly elevated. Serum cationic trypsinogen declined with age in patients with pancreatic insufficiency, reaching low or undetectable levels after 6 years. In contrast, serum anionic trypsinogen levels remained normal or elevated in 33% of those over 6 years of age. There was no age-related change in either cationic or anionic trypsinogen among the CF patients with pancreatic sufficiency, and the majority had normal or elevated levels. Serum samples from selected CF patients were separated into IgG and non-IgG fractions using Staph. Protein A columns. Immunoreactive cationic and anionic trypsinogen were detectable in the non-IgG fractions of sera from CF infants and older patients with pancreatic sufficiency. In older CF patients with undetectable serum cationic and anionic trypsinogen, no immunoreactive material was detectable in either the IgG or non-IgG fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Antibodies that are Specific for Human Carboxypeptidase B but not for the Zymogen Procarboxypeptidase B by Affinity Chromatography

A major theory for the pathogenesis of acute pancreatitis is that the normally inactive precursors of pancreatic proteases become activated, with subsequent destruction of enzymes, polypeptide hormones and other proteins in tissue and blood. To investigate the role of proteases in the disease, we developed radioimmunoassays for a number of these enzymes. However, experiments with serum samples fractionated by gel filtration showed that the amount of immunoreactive protein is not directly related to the amount of active enzyme against which the antiserum was directed. For example, procarboxypeptidase B effectively crossreacts about 30% with carboxypeptidase B using antiserum to active enzyme. To obtain antibodies that are specific for active enzyme, we treated the antiserum with resin containing immobilized procarboxypeptidase B. Stepwise addition of the resin produced antiserum with progressively less reactivity towards the zymogen relative to the enzyme, with the final material having less than 1% crossreactivity. The titer of enzyme-specific antiserum was 1% that of the original; however, it could be estimated that 10% of the original antibodies were specific for the active enzyme. To increase the yield of enzyme-specific antibodies, we are currently trying to obtain a mouse hybridoma that produces the desired antibody.