The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

Abstract

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4°C and prolonged preincubation at 25°C. Its activation often followed that of the procollagenase present in the same media. 3. Activation ofneutral proteinase (as does that ofprocollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes

Secretion of a latent neutral proteinase that degrades cartilage proteoglycans by skin and synovial fibroblasts in culture

“…It can thus be identi~ed as a neutral n~etal-depen~~ent proteinase that degrades the protein-core of the cartilage proteoglycan subunits. It appears similar to enzymes secreted by mouse bone explants [7] and rabbit macrophages [8] or extracted from human cartilage [ 101 but it differs from the proteoglycandegrading neutral setine-proteinases present in human ~~ly~~~~~~phonuc~~~r leucocyres f3,117_…”

“…Three days later, the cells were washed extensively and further cultured for 7 days in serum-free (unless otherwise indicated) DMEM. The conditioned culture media were then collected and activated with trypsin as in [6,7]. The proteoglycan-degrading neutral proteinase present in serum-free media was fully activated by preincubation for 10 min at 25°C with 8 pg trypsin/ml followed by the addition of a 4-fold excess of soya-bean trypsin inhibitor to block the action of trypsin.…”

“…The proteoglycan-degrading neutral proteinase present in serum-free media was fully activated by preincubation for 10 min at 25°C with 8 pg trypsin/ml followed by the addition of a 4-fold excess of soya-bean trypsin inhibitor to block the action of trypsin. It was assayed [7,8] either by measuring the amount of 35S-labelled fragments released at pH 7 from 35S-labelled rabbit ear cartilage or by following, at pH 7.4, the decrease in viscosity of a solution of proteoglycan subunits purified [9] from bovine nasal cartilage. One unit of activity refers to the amount ofenzyme that releases 1% initial cartilage 35S radioactivity/h.…”

“…One unit of activity refers to the amount ofenzyme that releases 1% initial cartilage 35S radioactivity/h. Gel filtration of the degradation products of 35S-labelled cartilage was done on Sepharose 6B as in [7] . All materials and chemicals were from the same suppliers as in [7,8].…”

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

“…Determination of cholesterol [35] did not reveal any preferential association with a particular fraction. A neutral proteinase as measured on [3H]acetyl casein [36] exhibited optimal activity at pH 6.5 in 0.1 M phosphate buffer; its sedimentation, latency and inhibition properties resembled those of acid proteinase.…”