A specific radioimmunoassay has been developed for human pancreatic cationic trypsin. The assay has been employed for the determination of immunoreactive forms of pancreatic cationic trypsin in blood. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone (TLCK) to prevent binding of the tracer to the serum inhibitors while maintaining its immunoreactivity. The average normal serum level determined was 26 ng/ml, with a range of 12--41 ng/ml. Eight of nine patients with acute pancreatic inflammation had at least a 15-fold elevation of total serum immunoreactive cationic trypsin. Cationic trypsinogen and cationic trypsin bound to alpha1-antitrypsin cross-react strongly in the radioimmunoassay. Thus it is possible to measure these potential molecular forms of cationic trypsin in serum. When normal human serum was fractionated on Sephadex G-200, all of the immunoreactive material eluted as a single peak of approximately 23,000 mol wt. No cationic trypsin could be detected in association with alpha1-antitrypsin or alpha2-macroglobulin. The 23,000-mol-wt peak was definitively shown to contain trypsinogen by affinity chromatography and by activation with human enteropeptidase. The identification of cationic trypsinogen in blood implies that the zymogen is secreted into the circulation by the pancreas rather than entering the bloodstream via absorption from the intestine.

Enteropeptidase↗

PubMed1979

Molecular forms of immunoreactive pancreatic cationic trypsin in pancreatitis patient sera.

The molecular forms of immunoreactive pancreatic cationic trypsin in sera of patients with acute pancreatic inflammation have been characterized using a radioimmunoassay technique that is capable of detecting trypsinogen as well as trypsin bound to alpha 1-antitrypsin. Trypsin bound to alpha 2-macroglobulin is not immunoreactive under normal assay conditions. However, alpha 2-macroglobulin-bound trypsin can be detected after gel filtration of serum on Bio-Gel A-0.5 m and acid treatment of column fractions. The average serum level of immunoreactive cationic trypsin from 20 patients with acute pancreatic inflammation was 1,590 ng/ml. An average normal value of 26 ng/ml has been obtained previously. Serum samples from 14 patients with pancreatic inflammation were chromatographed under conditions that resolve trypsinogen, alpha 1-antitrypsin-bound trypsin, and alpha 2-macroglobulin-bound trypsin. In each case, the major portion of the immunoreactive material eluted at a position corresponding to free trypsinogen, while a minor fraction of the immunoreactive material appeared to be trypsin bound to alpha 1-antitrypsin. The zymogen nature of the major peak was confirmed in one case by activation with human enteropeptidase. In 11 of 14 patients, acid treatment of the alpha 2-macroglobulin peak yielded immunoreactive trypsin.

Cations↗

PubMed1978

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay.

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors alpha1-antitrypsin and alpha2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.

Anions↗

PubMed1978

Structural basis for the specific activation of human enteropeptidase.

Human cationic trypsinogen is activated by human enteropeptidase much more readily than bovine trypsinogen, the ratios kcat/Km being 330 and 11 mM-1S-1, respectively. Conversely, porcine enteropeptidase activates bovine trypsinogen much more rapidly (kcat/Km = 630 mM-1S-1) than human cationic trypsinogen (kcat/Km = 2.4 mM-1S-1). The primary structure of the activation region of human cationic trypsinogen has been investigated in an attempt to elucidate the basis for these findings. The sequence of the first 12 residues at the NH2-terminus of human cationic trypsinogen has been shown to be Asp-Lys-Ile-Val-Gly-Gly-Tyr-Asn-Cys-Glu-Glu-Asn. Furthermore, the activation peptide derived from human cationic trypsinogen has been isolated and shown to be the dipeptide Asp-Lys. This result is in contrast to the Val-(Asp)4-Lys activation peptide from bovine trypsinogen and demonstrates that human cationic trypsinogen does not contain the (Asp)4 sequence present in many other mammalian trypsinogens. It is proposed that the high degree of specificity for activation of human cationic trypsinogen by human enteropeptidase is due to the preferential recognition of the novel activation peptide sequence in the human zymogen. Thus, these two functionally related proteins, cationic trypsinogen and enteropeptidase, may have evolved in a parallel manner in the human lineage.

Amino Acid Sequence↗

PubMed1978

Human cationic trypsinogen. Purification, characterization, and characteristics of autoactivation.

Human pancreatic cationic trypsinogen has been purified to homogenity from an acetone powder of pancreatic tissue. After an initial ion exchange chromatography step on sulfopropyl (SP)-Sephadex at pH 2.6, cationic trypsinogen was separated from the majority of trypsin activity by passage through an affinity column of lima bean trypsin inhibitor-agarose at high ionic strength. The zymogen was then further purified by affinity chromatography on the same material at low ionic strength. Highly purified trypsinogen was resolved from containing chymotrypsinogen by ion exchange chromatography on SP-Sephadex at pH 6.0. The purified zymogen was shown to be homogeneous by polyacrylamide gel electrophoresis at pH 2.1 and at pH 4.3 as well as by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoactivation of human trypsinogen was investigated at pH 5.6 and at pH 8.0. The rate of autoactivation of the human zymogen is rapid at pH 5.6 and is maximal in approximately 1 mM Ca2+. These results are in marked contrast to those previously reported for autoactivation of bovine trypsinogen, which is extremely slow at pH 5.6 and which shows a dependence on at least 50 mM Ca2+ for maximum rate of activation (MacDonald, M. R., AND Kunitz, M. (1941) J. Gen. Physiol. 25, 53-73).

Calcium↗

PubMed1978

Studies on the ascites fluid of acute pancreatitis in man.

Total protein, alpha1-antitrypsin, alpha2-macroglobulin, amylase, methemalbumin, tryptic amidase activity, radioimmunoassayable elastase 2, and three lysosomal hydrolases were determined in the ascites fluid from patients with acute pancreatitis. In eight patients methemalbumin was detected in ascites and serum, supporting the diagnosis of hemorrhagic pancreatitis. Significant levels (4-45 microgram/ml) of tryptic amidase activity were detected in ascites samples from all patients. Evidence is presented which demonstrates that the tryptic amidase activity is due to alpha2-macroglobulin-bound trypsin. Pancreatic elastase 2, determined with a new sensitive and specific radioimmunoassay, ranged from 400 to 2100 ng/ml in serum and from 650 to 4460 ng/ml in ascites fluid. Substantial amounts of alpha2-macroglobulin-bound trypsin and elastase 2, entering the circulation from the peritoneal cavity, might be responsible for certain serious complications seen in acute pancreatitis. However, with the exception of serum calcium and methemalbumin and the ascites fluid methemalbumin and total protein, none of the biochemical parameters studied showed a distinct correlation with the patient's outcome.

Acute Disease↗

PubMed1977

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

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Proinsulin↗

PubMed1977

The stoichiometry of inhibition of human pancreatic elastase 2 by human alpha1-antitrypsin.

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Binding Sites↗

PubMed1977

Pancreatic elastase in human serum. Determination by radioimmunoassay.

This study demonstrates that a serine endopeptidase of pancreatic origin (elastase 2) circulates in human blood. A specific and highly sensitive radioimmunoassay has been developed for pancreatic elastase 2 in human serum. The inactivation of elastase 2 employed as radioiodinated tracer with an active site-specific reagent (phenylmethanesulfonyl fluoride) was necessary to prevent its binding by serum alpha1-antitrypsin and alpha2-macroglobulin while maintaining its immunoreactivity. The assay is based upon competition of standard human pancreatic elastase 2 with 125I-labeled phenylmethanesulfonyl elastase 2 for specific antibody binding sites, after which a second antibody precipitation step is used to separate bound from free 125I-labeled phenylmethanesulfonyl elastase 2. The minimum detectable concentration of elastase 2 was 0.9 ng/ml. The average normal fasting serum level determined was 71 ng/ml, approximately 80-fold greater than the minimum detectable amount. The form of radioimmunoassayable elastase 2 in normal human serum has been investigated by gel filtration of serum samples on Sephadex G-200 followed by radioimmunoassay of column fractions. The majority of the immunoreactive elastase 2 is eluted from G-200 in the void volume. While a minor amount of elastase 2 is eluted in a position consistent with alpha1-antitrypsin-elastase 2 complex, no free elastase or free proelastase is detectable. Addition of exogenous elastase 2 to normal serum prior to gel filtration on G-200 produced an increase only in the peak of radioimmunoassayable elastase bound to alpha1-antitrypsin. In vitro experiments have demonstrated that while elastase 2 bound to alpha1-antitrypsin is immunologically reactive, alpha2-macroglobulin-bound elastase 2 cross-reacts less than 2% in this radioimmunoassay. The assay has been shown to be specific for elastase 2. Human pancreatic elastase 1, anionic trypsin, chymotrypsin I, and chymotrypsin II do not cross-react in this assay system. The major advantages of this radioimmunoassay over enzymatic assays are its high sensitivity and ability to measure the enzyme in terms of its total protein concentration.

Cross Reactions↗

PubMed1976

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.

Amino Acids↗

PubMed1976

Purification and characterization of two human pancreatic elastases.

Two proteases with elastolytic activity (elastases 1 and 2) have been isolated from activated extracts of human pancreatic tissue. The purification procedure for both elastases included ammonium sulfate fractionation followed by ion-exchange chromatography on CM-Sephadex C-50. Elastase 1 was further purified by chromatography on DEAE-Sephadex A-50. The homogeneity of both enzymes was demonstrated by Sephadex G-75 gel filtration, analytical polyacrylamide disc gel electrophoresis at pH 2.3, 4.5, and 8.3, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3. Both enzymes hydrolyzed undyed elastin as well as Remazol brilliant blue elastin and Congo red elastin. Activities and kinetic parameters using several synthetic substrates are also reported. The enzymes were further characterized in terms of molecular weight, amino acid composition, and N-terminal and penultimate amino acid residues. Their inhibition by the human serum protease inhibitors alpha2-macroglobulin and alpha1-antitrypsin was also studied. Elastase 1 appears to be very similar to human protease E (Mallory, P. A., and Travis, J. (1975), Biochemistry 14, 722). Elastase 2 is distinct from all human pancreatic proteases which have been characterized to date.

Amino Acid Sequence↗

PubMed1975

Human pancreatic carboxypeptidase B. I. Isolation, purification, and characterization of fraction II.

Human carboxypeptidase B fraction II has been purified from pancreatic juice by DEAE-'Sephadex' chromatography, isoelectric focusing, and 'Sephadex' G-100 gel filtration. The enzyme has been characterized by analytical polyacrylamide disc-gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis Km determination, molecular weight determination on 'Sephadex' G-100, zinc analysis, and inhibition by metal chelating agents. Human carboxypeptidase B fraction II appeared homogeneous in analytical polyacrylamide disc-gel electrophoresis, but showed two components of 23,500 and 9,200 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Zinc analysis revealed 0.96 gram atoms of zinc per mole of enzyme, and a Km of 65 +/- 3 muM was determined for hydrolysis of hippuryl-L-arginine.

Amino Acids↗