The primary structure of a pig leucocyte cysteine proteinase inhibitor, also called cathelin, was determined. The sequence was obtained from analyses of peptides isolated from the chymotryptic, endoproteinase Lys-C and protease V8 digests, and by analysis of the peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The inhibitor consists of 96 residues. The N-terminal residue of the inhibitor is pyrrolidonecarboxylic acid. The amino acid sequence. of cathelin suggests the appearance of a new family of cysteine proteinase inhibitors.

The amino acid sequence of a cathepsm D mhibitor tsolated from potato is describedIt was determmed by analysis of peptides generated by use of the glycine-spectfic proteinase PPIV. The order of the peptides was established by exammation of tryptic peptides derived from the two cyanogen bromide peptides The Inhibitor comprises 187 ammo acid restdues. and has a calculated M, of 20 450Aspartic protemase mhtbitor, Ammo acid sequence: Cathepsm D

A protein inhibitor of cysteine proteinases, "stefin", was purified from cytosol of human polymorphonuclear granulocytes. Affinity chromatography on carboxymethylated papain-Sepharose was used as the first step, followed by ion exchange chromatography on DEAE-Sephacel, which resolved four inhibitory peaks. The main peak, comprising approx. 80% of total inhibitory activity was characterized.It was found to be a homogenous protein with an apparent molecular mass slightly lower than that of cytochrome c and with an isoelectric point of 4.65. The inhibitor inhibits papain at a molar ratio of 1:1 as well as cathepsin B and H, but it does not inhibit serine and aspartic proteinases. It is stable at elevated temperature and in alkaline pH, but looses its activity in acid pH. Oxidized glutathione has no effect on its inhibitory activity. Cysteinproteinasen-Inhibitoren, L Isolierung und Charakterisierung des Cysteinproteinasen-Inhibitors Stefin aus dem Cytosol menschlicher polymorphkerniger GranulozytenZusammenfassung: Aus dem Cytosol menschlicher polymorphkerniger Granulozyten wurde der Cysteinproteinasen-Inhibitor Stefin isoliert. Nach Affinitätschromatographie an carboxymethylierter Papain-Sepharose wurden in einem zweiten Schritt durch lonenaustauschchromatographie an DEAE-Sephacel vier Inhibitorfraktionen getrennt. Die Hauptfraktion, die 80% der gesamten Inhibitoraktivität enthält, wurde näher charakterisiert.Es handelt sich um ein einheitliches Protein vom isoelektrischen Punkt 4.65, dessen apparente Molekularmasse etwas unter der von Cytochrom c liegt. Der Inhibitor hemmt Papain in einem molaren Verhältnis von l: l sowie Cathepsin B und H, jedoch nicht Serin-und Asparaginsäure-Proteinasen. Er ist bei erhöhter Temperatur und alkalischem pH stabil, verliert jedoch im Sauren rasch seine Aktivität. Oxidiertes Glutathion beeinflußt die Inhibitoraktivität nicht.

A new low-molecular mass cysteine molecular mass of approximately 12 kDa and a pi of proteinase inhibitor (CPI) was purified from the 4.8. The amino terminus is blocked. The amino-acid cytosol of peripheral pig leukocytes. The isolation composition and the sequence of the C-terminal part procedure included DEAE chromatography, Seof the molecule are suggestive of a new family of phadex G-100 gel filtration and fast-protein liquid cystatins. The CPI was found to be a tight-binding chromatography on Mono Q. The procedure resulted inhibitor of both papain and cathepsin L, with K\ in the isolation of a homogenous protein with a values of O.lnM and InM, respectively. Ein neuer Typ eines niedermolekularen Cystein-Proteinase-Inhibitors aus SchweineleukozytenZusammenfassung: Ein neuer Cystein-ProteinaseMolmasse von 12 kDa und einem p/von 4.8 isoliert. Inhibitor (CPI) von niedriger Molekularmasse wurde Sein Aminoterminus ist blockiert. Die Aminos ureaus Cytosol von Schweine-Leukozyten isoliert. Das Zusammensetzung und eine partielle Sequenz des Reinigungsverfahren schlie t DEAE-ChromatograCarboxylterminus deuten auf eine neue Familie der phie, Gelfiltration ber Sephadex G-100 und FPLC Cystatine hin. Der CPI ist ein sehr fest bindender an Mono Q ein. Mit der beschriebenen Methode Inhibitor f r Papain (K, = O.lnM) und Cathepsin L wurde ein homogenes Mini-Protein mit einer (K t = InM).

SummaryA plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pi of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37° C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.