James W. Brodrick

Proinsulin conversion to desalanyl insulin by alpha2-macroglobin-bound trypsin.

HARPEL and Mosesson1 observed that α2-macroglobulin-bound trypsin hydrolysed low molecular weight ester and amide substrates at rates comparable with those of free protease, but degradation of high molecular weight proteins was markedly inhibited. To account for this finding, Barrett and Starkey2 introduced the novel concept of steric entrapment of endopeptidases by α2-macroglobulin. Neurath and Walsh3 have recently reviewed the role of proteolytic enzymes in the conversion of a variety of enzymes, hormones, and other physiologically active proteins from inactive precursors to active forms by limited proteolysis. Many of these physiologically active proteins exist in the bloodstream in both precursor and active forms. We report here that proinsulin, a large polypeptide of 9,000 molecular weight, is rapidly hydrolysed by α2-macroglobulin-bound trypsin. This finding suggests that pro-teases bound to α2-macroglobulin may be involved in limited proteolytic cleavage associated with activation of precursor molecules as well as the degradation of biologically active polypeptides in the circulation.

[17] Radioimmunoassay determination of circulating pancreatic endopeptidases

Publisher Summary This chapter describes the radioimmunoassay determination of circulating pancreatic endopeptidases. The radioimmunoassay technique has been applied to the detection of pancreatic endopeptidases in plasma and serum. These studies have demonstrated that, while the bulk of the digestive enzymes enter the pancreatic ductal system, a small fraction enters the bloodstream. The application of the radioimmunoassay methodology has permitted the characterization of the circulating molecular forms of human cationic trypsin, anionic trypsin, elastase 2, and chymotrypsin. The ability to quantitate the levels of total immunoreactive trypsin in biological fluids as well as to identify the molecular forms of trypsin present in the sample has resulted in several reports of clinical applications for the radioimmunoassay of cationic trypsin. The lack of cross-reactivity in the radioimmunoassay of a given pancreatic endopeptidase, with the same protease, from other mammalian species, demonstrates that a separate radioimmunoassay must be developed for each enzyme to be investigated. The lack of cross-reactivity between the various pancreatic endopeptidases of the same species permits the specific identification of each protease in a biological sample.

Plasma immunoreactive pancreatic cationic trypsinogen in cystic fibrosis: a sensitive indicator of exocrine pancreatic dysfunction.

Summary: Plasma immunoreactive cationic trypsin(ogen) levels were determined in 32 control subjects and 43 patients with varying degrees of pancreatic insufficiency including 35 with cystic fibrosis (CF) and eight with Shwachmans syndrome. In six CF infants less than 2 years of age, plasma trypsin(ogen) levels were significantly elevated (97.3 ± 62.2 ng/ml) above the normal range for nine controls (7.0 ± 5.9 ng/ml; P < 0.025). Four of these infants had steatorrhea, three of whom had undetectable duodenal trypsin activity after stimulation with secretin-cholecystokinin. In two CF infants, molecular size fractionation by gel filtration of plasma followed by radioimmunoassay of the column fractions demonstrated that trypsinogen was the only immunoreactive species in the circulation.In contrast, in older CF patients with steatorrhea (mean age, 15.3 ± 4.6 years), plasma cationic trypsin(ogen) levels were undetectable or low (1.1 ± 1.7 ng/ml). This finding clearly distinguished them from older CF patients without steatorrhea (mean age, 14.3 ± 3.9 years) in whom cationic trypsin(ogen) levels were significantly higher (23.3 ± 17.6 ng/ml; P < 0.01). The mean trypsin(ogen) concentration in the older CF patients without steatorrhea did not differ from the mean value for 23 normal subjects of similar age. Plasma cationic trypsin(ogen) levels in two Shwachmans patients with steatorrhea (0.19 and 0.86 ng/ml) were significantly lower than the values found in six Shwachmans patients without steatorrhea (5.9 ± 2.3 ng/ml; P < 0.025). Furthermore, in nine older CF patients and eight Shwachmans patients, circulating trypsin(ogen) levels were highly correlated with duodenal trypsin output after secretin-cholecystokinin stimulation (r = 0.946, P < 0.01; r = 0.899, P < 0.01, respectively). These results suggest that in CF infants high levels of circulating trypsin(ogen) persist even in those with complete pancreatic insufficiency. In older CF patients and those with Shwachmans syndrome, however, circulating trypsin(ogen) accurately reflects residual pancreatic function.Speculation: Cystic fibrosis (CF) infants often possess viable but ductally obstructed pancreatic tissue, which may be destroyed with disease progression. The correlation between pancreatic exocrine function and circulating trypsin(ogen) in older CF patients and Shwachmans patients, however, indicates that ductal obstruction is not a prominent feature in these patients.

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.

542 CIRCULATING IMMUNOREACTIVE PANCREATIC CATIONIC AND ANIONIC TRYPSIN(OGEN) IN PANCREATIC INSUFFICIENCY

Two normal variants of trypsin(ogen), cationic (CATRYP) and anionic (ANTRYP) trypsin(ogen), can be identified by the immunoassay technique. In normal subjects CATRYP exceeds ANTRYP by approximately 4:1 in both pancreatic secretions and in the peripheral circulation. We measured random serum CATRYP and ANTRYP in 19 patients with varying degrees of pancreatic insufficiency, including 8 with Shwachmans syndrome (SHW) and 11 CF patients (> 3 years of age). Total duodenal tryptic activity had been determined by quantitative collection of duodenal secretions following stimulation with cholecystokinin/secretin. There was a high degree of correlation between serum CATRYP and total duodenal tryptic activity in both SHW (r = 0.90) and CF patients (r = 0.93). There was no correlation, however, between serum ANTRYP and duodenal tryptic activity (SHW; r = 0.46: CF; r = 0.07) due to persistence of serum ANTRYP in 5/8 patients with < 5% of normal pancreatic function. In contrast, duodenal juice samples failed to show persistence of ANTRYP and both CATRYP and ANTRYP were diminished proportionately in a ratio of 4:1. Unlike CATRYP, serum ANTRYP is a poor index of pancreatic function, due to the apparent persistence of this material in the serum of patients with severe pancreatic insufficiency.