Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

Abstract

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose.Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAEcellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, a,-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used. The third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, a,-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%.In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-1 00 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80 %) of post-granular supernatant was eluted together with ovalbumin ( M , 43000) and the remainder with cytochrome c (12 300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase.Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate.Inhibitors of post-granular supernatant are stable at pH 6 -8, but unstable in the pH range 2 -5 and are thermolabile.Neutral tissue and cell proteinases have been rather poorly investigated in comparison to acid proteinases. The reason is that it was not possible to prepare stable active samples. A higher neutral proteinase activity was obtained only from granule (lysosomal) extracts [l -31, or from so-called reextracts, where the first cell extract was discarded; i.e. only from samples where the cytoplasm had in any case been removed [4,5].The last investigations in this field have shown that besides neutral proteinases inhibitors of these proteinases are also present in the cells and that for a successful isolation of neutral proteinases these inhibitors must be removed first. There are no data concerning the presence of any well-characterized material from animal tissues capable of inhibiting intracellular acid proteinases, such as cathepsin D [6].Davies and coworkers [7] reported in their study of neutral proteinases of polymorphonuclear cells that cytoplasmic -postgranular supernatant fraction caused the strongest inhibition of neutral proteinases. Because the polymorphonuclear cells were obtained from inflammatory exudates, they proposed a ...

Human‐Polymorphonuclear‐Leucocyte Neutral Protease and Its Inhibitor

“…The granule fraction contained a neutral protease capable of attacking the telopeptide regions in fluorescein-labelled polymeric collagen fibrils, confirming earlier reports [1,2]. When 100 pl of the granule extract was incubated with the fluorescent substrate in the presence of increasing quantities of the postgranule supernatant fraction for 1.5 h (Fig.…”

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes

“…Preliminary observations suggested that the cytosol of neutrophils contains inhibitors of the collagenolytic activity of granule extracts (Kopitar & Lebez, 1975);…”

Isolation and Some Characteristics of Urokinase Inhibitors Isolated from Pig Leucocytes

“…Leucocytes were isolated from pig blood (5,400 ml), as described previously (17). The cells were suspended in 0.34 M sucrose and homogenized with a Teflon pestle.…”

“…In the course of our continuing studies, two types of specific inhibi tors of proteinases were isolated from pig leucocytes and from bovine spleen. These inhibitors are acidic proteins (Ip 4.6-5.6) and differ in molecular weight, inhibitory ability against tested enzymes and immunologically (4,(17)(18)(19)(20)(21). Neither Inhibitor-1 (1-1) nor Inhibitor-2 (1-2) inhibits trypsin or urokinase.…”

“…Leucocyte cells have been shown to contain inhibitory activity against elastases, chymotrypsin-like neutral proteinases, thiol proteinases and trypsin (7,9,16,17,31). In the course of our continuing studies, two types of specific inhibi tors of proteinases were isolated from pig leucocytes and from bovine spleen.…”