Date of Award
5-1971
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Medical
First Advisor
Beatrice Kassell
Second Advisor
Harris L. Friedman
Third Advisor
Alan H. Mehler
Fourth Advisor
Samuel A. Morell
Fifth Advisor
Fumito Taketa
Abstract
Pepsins, the digestive enzymes of the stomach, are synthesized as inactive zymogens. These are secreted by the gastric mucosa in multiple forms in man and other species and are converted to active enzymes by the acid conditions of the stomach. Information regarding peptic enzymes has been gained mainly from studies on the pepsinogens and pepsins from pig stomach. Studies on homologous enzymes of different species can aid in identifying the parts of these molecules essential for activity and for maintenance of conformation.
This investigation is concerned with the structure and properties of several pepsins derived from bovine fundic mucosa. Highly purified pepsin was prepared by isolating the precursor and converting it to the active enzyme according to procedures developed in this laboratory. The pepsin was separated into four components by chromatography on hydroxylapatite. The amino acid compositions of the bovine pepsins do not differ among themselves, but differences previously found in organic phosphate content are confirmed. Bovine pepsin shows similarity in composition to porcine and human pepsins with respect to the large number of acidic and small number of basic residues. However, the content of the individual amino acids varies considerably among these pepsins. Bovine pepsin, l ike human pepsin, has no lysine.
Kinetic studies were carried out on bovine and porcine pepsins, using several synthetic substrates. The bovine pepsins do not differ from each other in activity, showing that the phosphate content has no effect on the activity with any of the substrates tested. Comparative studies with porcine pepsin showed rather marked quantitative differences, although the two species have similar pH optima. Bovine pepsins have about 25% and 40% of the activity of porcine pep si n toward N-acetyl-Lphenylalanyl- L-diiodotyrosine and N-acetyl-L-phenylalanyl-L-tyrosine, respectively. Bovine pepsins showed very little activity toward benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tryptophan ethyl ester, a good substrate for porcine pepsin. Bovine pepsins have 60 to 70% of the activity of porcine pepsin with hemoglobin as substrate. Bovine and porcine pepsins have similar milk clotting activity.
Bovine pepsin appears to be more resistant than porcine pepsin to the denaturation that occurs with increase in pH. Up to pH 6, both species are stable. At pH 7, the bovine species retains 70% of its activity under the same conditions in which the porcine species is completely inactivated. The differences in catalytic activity found between bovine and porcine pepsin cannot be correlated with differences in amino acid composition at this time. However, as sequence determinations become available on bovine pepsins (studies now underway in this laboratory), these kinetic studies will contribute to the understanding of structure-function relationship.