Barbara J. Allan

Human pancreatic proteins: Amylase, proelastase, and trypsinogen

Abstract Alpha-amylase exists in human pancreatic juice in multiple molecular forms. Up to six isoenzymes have been demonstrated by polyacrylamide disc gel electrophoresis and starch-digestion zymograms. Five of the isoenzymes account for most of the amylase present, but analysis of higher amylase loads reveals the sixth form. The isoenzymes are not artefacts due to proteolysis nor to photocatalyzed polymerization of sample gels prior to electrophoresis. Comparison of the isoenzymes of human pancreatic amylase with those of human parotid amylase on polyacrylamide disc gels shows no common bands. Both human amylases are retarded on columns of Bio-Gel P-150 beyond the elution volume expected for molecules of their reported sizes, the pancreatic enzyme being held slightly longer than the parotid enzyme. Proelastase has been demonstrated among the more basic proteins. The proelastase is activated by bovine trypsin to a specific enzymatic activity approximately onesixth that of crystalline porcine elastase. A pro-ATEEase is closely associated with the proelastase fraction. A human trypsinogen was isolated from the anionic protein fraction. It can be activated by bovine trypsin, porcine enterokinase, or spontaneously during prolonged incubations. The specific enzymatic activity of the human trypsin studied is lower than that of the bovine enzyme.

Alteration of human pancreatic secretions by pancreatic and ampullary carcinoma. A computer-aided analysis of isoelectric focusing patterns

Abstract Human pancreatic secretions obtained from patients with pancreatic carcinoma and carcinoma of the ampulla of Vater differ significantly from those obtained from patients without pancreatic disease. Differences were also apparent between secretions from the two types of carcinoma cited above. This was shown by computer analysis of the isoelectric focusing protein patterns from 30 specimens. Average densitometric tracings ± the standard deviation were prepared by the computer for the acidic portion (pH 3–5.7) of the pH 3–10 isoelectric focusing patterns for each group of ten specimens. In this pH region 19 bands were located by the computer. The major bands had isoelectric points of 4.7, 5.0, 5.2, and 5.7.

Techniques for reproducible transient-state isoelectric focusing of human pancreatic secretory proteins with computer-assisted pattern matching, averaging, and analysis

Abstract A new technique is described for obtaining reproducible transient-state isoelectric focusing patterns of human pancreatic secretory proteins. This method is excellent for preservation of the proteins for further analysis because of the avoidance of urea, the short running times, and low temperatures. The protein patterns were analyzed on a point-by-point basis by a computer using an extension of our previously published method (Allan, B. J., Kirk, J., and White, T. T., 1978, Biochem. Biophys. Res. Commun. , 85 , 1239–1246). It was possible with these techniques to subtract various types of backgrounds and to construct average densitometric tracings for statistical analysis.

A refined cellulose acetate electrophoretic technique for analysis of human pancreatic juice

Abstract Fourteen to 18 protein bands were found to be present in inactive human ductal pancreatic juice by a cellulose acetate electrophoretic technique. Lyophilization of the samples did not alter the patterns significantly. Quantification of the protein bands was possible by scanning densitometry. Intensity of the protein bands was proportional to the protein concentration of the sample applied. Amylase, lipase, two ribonucleases, two procarboxypeptidases A, one proelastase, two trypsinogens, and two major and two to four minor chymotrypsinogens (in some specimens) were detected histochemically. Active ductal pancreatic juice contained seven distinct and several minor bands. Trypsin, chymotrypsin, elastase, amylase, and carboxypeptidase A bands have been identified. A dark band at the origin, which was a precipitate removable by centrifugation, was also found in all active specimens. When benzamidine was used, the migration rate of the trypsin band was altered. Densitometric scans of protein patterns after cellulose acetate electrophoresis and band identification by histochemical methods gave comparable results with DEAE elution diagrams obtained with classical biochemical methods (Fig. 5).

Tibias—A microcomputer-based analysis system for isoelectric focusing and electrophoretic patterns

A program is described for a computer-aided analysis system for isoelectric focusing (IEF) patterns. This system is primarily aimed at comparing and statistically analyzing large numbers of one-dimensional electrofocusing patterns in the search for specific tumor markers in pancreatic ductal secretions. It is designed to collect raw densitometric data, allow interactive processing, matching and statistical tests, and prepare high-quality plots of data at various stages.

Protein Secretions in Hamsters with Pancreatic Carcinoma

Pancreatic secretions from the hamster model for pancreatic ductal adenocarcinoma were analyzed to determine whether alterations had occurred in the protein composition. CCK- and secretin-stimulated secretions were collected from 13 animals with cancer and 16 normal controls. Subsequent separation of the proteins by isoelectric focusing showed the following: (1) Significant changes occurred in the protein composition from animals with carcinoma. An unusually dark band was present at pH 7.5 just below amylase (pH 7.65); two unidentified bands, present in the normals at pH 6.9 and 7.0, were missing; and marked decreases occurred in the cathodic proteins with isoelectric points above pH 9. (2) CCK provided the optimal stimulus for differentiating specimens from animals with carcinoma from the normal controls. (3) The protein concentrations of CCK-stimulated secretions of animals with carcinoma were significantly lower than the controls. We have concluded that the protein alterations which have occurred in the hamster model for pancreatic ductal adenocarcinoma warrant further investigation.

Autonomic regulation of potassium release from human labial salivary glands in vitro.

Labial salivary-gland slices from normal human subjects were incubated in vitro according to various protocols. The autonomic regulation of these was significantly different from that of most major salivary glands. K release was stimulated by incubation with a cholinergic agonist but not with alpha- or beta-adrenergic agonists. The cholinergically-induced K release was dependent on agonist concentration and was inhibited by the addition of atropine, and by the removal of Ca in the presence of EGTA.

Human pancreatic secretory protein profiles in the search for tumor markers

Pancreatic cancer secretory protein profiles were shown to be different from normals by a microcomputer-assisted analysis of isoelectric focusing patterns. Two acidic protein band regions (pI 3.0 and 4.5) of the pancreatic carcinoma profiles were significantly increased, and eight protein bands (pI 3.7, 5.0, 5.5, 6.5, 6.9, 7.3, 8.0, >10.0) were significantly decreased. Highly significant decreases occurred at pH 7.3 (chymotrypsinogen) and at pH 5.0 (procarboxypeptidase Al, DNase I) in nonactivated specimens. The ratio between the absorbance points at 2.08 and 2.63 cm from the anode in each protein pattern differentiated the pancreas cancer specimens from the control group. Profiles found for the control group gave pI values similar to those found in the literature. The potential value of these findings in the search for tumor markers warrants further investigation as to whether these specimens can be differentiated from those from chronic pancreatitis patients.

Human pancreatic secretory protein profiles in pancreas cancer and chronic pancreatitis

Pancreatic secretory protein profiles differed significantly between patients with chronic pancreatitis (CP) and pancreatic carcinoma (CA). Specific regions of the patterns were altered when CP versus CA and when CP versus normals were compared. Bands isoelectric in the region of pH 9–11 were elevated in CP. The possible identification of this band as lactoferrin is discussed.

Bile, pancreatic cancer, and the activation of pancreatic juice.

Abstract Bile and pancreatic juice vary in their trypsin inhibitor content. Activation of bile-pancreatic juice mixtures (1:1) occurred when the combined trypsin inhibitory capacity was low. No activation was seen with bile having a high trypsin inhibitor content. Pancreatic juice activated faster where more trypsin inhibitor complex was present. With calcium (25 mM), pancreatic secretions activated in a similar order as with the trypsin-inhibitor-free bile. We propose that activation occurs more readily where large amounts of the trypsin-inhibitor complex are present in pancreatic juice as a result of carcinoma or other etiologies.