Date of Award:

5-1983

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Committee Chair(s)

D. P. Cornforth

Committee

D. P. Cornforth

Committee

C. A. Ernstrom

Committee

R. J. Brown

Committee

A. W. Mahoney

Committee

T. M. Farley

Abstract

Cathepsin D has been postulated to play a role in the tenderization of meat with ageing. Meat toughness has been related to connective tissue content and degree of myofibrillar shortening. This investigation was undertaken to determine the activity of cathepsin D on bovine intramuscular connective tissue.

Highly purified cathepsin D preparations from bovine spleen and bovine sternomandibularis muscle were obtained by ammonium sulfate fractionation followed by affinity chromatography on pepstatin coupled to controlled pore size alkyl amine glass beads. Maximum proteolytic activity using hemoglobin as substrate was obtained at pH 3.5 and 3.8 for bovine spleen and muscle cathepsin D, respectively. The banding pattern of bovine spleen cathepsin D on SDS-polyacrylamide gels was identical to that of a commercial preparation. Both cathepsin D preparations had 2 major proteins with molecular weights of about 45,000 and 35,000 daltons. Bovine muscle cathepsin D preparations had 4 polypeptide bands corresponding to molecular weights of 57,000, 45,000, 41,000, and 20,000 on SDS gels. The 45,000 and 41,000 dalton components of muscle cathepsin D were shown to undergo degradation to release peptides of about 20,000 daltons during refrigerated storage of the 40-70 % ammonium sulfate fraction.

Cathepsin D from neither of the tissues had proteolytic action towards intramuscular connective tissue isolated from bovine shank muscle over the pH range 3.0- 4.5 following incubation for 24 h at 37 C. However, a commercial preparation of pineapple stem bromelain was found to degrade connective tissue, resulting in a release of 44,000 and 64,000 dalton peptides. Degradation was maximum at pH 3.0 and the rate of degradation decreased with pH increase. The failure of a highly purified cathepsin D preparation to degrade connective tissue is a clear indication that the meat tenderizing effect of cathepsin D during meat ageing is not due to its proteolytic action on intramuscular collagen, but due to its reported proteolytic action towards myofibrillar proteins.

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