Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin c) related to the commercial source of fetal bovine serum in the culture medium

Abstract

The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P less than 0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, beta-glucosidase, beta-glucuronidase, and N-acetyl-beta-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera.

Quantification of the hemolysis associated with use of T-61Ras a euthanasia agent in rabbits–a comparison with EuthanylR(pentobarbital sodium) and the impact on serum hexosaminidase measurements

“…The activity of hexosaminidase (Cabezas 1989) in the semm was determined following a previously published protocol (Doughty et al 1985;Doughty and Gmenstein 1988) using 4-methyl~kBliferyl-N-acety1-~-~)-gdactos~~de (M-9 129, Sigma) at pH 4.6 and 3'7°C. For the assays, 100 pL of a buffer solution (50 mM dimethylglutaric acid -NaOB; pH 4.6) was mixed with 100 pL of substrate solution (0.5 mM prepared in water), and 25 pL semm was added to start the reaction.…”

Assessment of Variability in Some Lysosomal and Soluble Enzyme Activities in Corneal Epithelium from Slaughterhouse Bovine Eyes

“…The pellet (Pio.ooo; the crude mitochondriallysosome fraction) was resuspended in 1.5 ml of homo genisation buffer. Enzyme assays were performed in duplicate at 37°C for 30 min for hexosaminidase us ing 0.4 mM 4-methylumbelliferyl-N-acetyl-P-Z)-galactosaminide at pH 4.6 in 250 mM sucrose [6], acid phosphatase using 10 m.M /Miitrophcnyl phosphate at pH 4.0 in 250 mM sucrose [7], lactate dehydrogenase using 2 mV/ DL-lactate and 5 m M p-NADH at pH 7 [8] and leucine arylamidasc using 0.06 m M leucyl-2-naphthylamide at pH 7.5 in the presence of 0.1 mM C0CI2 [9]. The lysosomal enzyme activities in the Pio.ooo material were assessed with and without Triton X-100 detergent 0.02% w/v to assess latency [6]; the concentration of Triton X-100 was determined, from dose-response curves, to be non-inhibitory.…”