Thermostable Endogenous Inhibitors of Cathepsins B and H

Abstract

Separate thermostable inhibitors of cathepsins B and H (IB and 1 , ) were found in every rat and human tissue that was analyzed. The inhibitors were separated from the cathepsins by heating at 80-90 "C at pH 3. I B and I H from rat lung were purified and found to occur in multiple molecular forms. Rat lung IB and IH were very similar proteins with molecular weights of 14000. 1s and I H from hog kidney were also purified and IH was obtained free of IB. These inhibitors also displayed microheterogeneity and had molecular weights of 11000. Hog kidney IH inhibited chymopapain, but did not inhibit bromelain, chymotrypsin or cathepsin D. Rat and human serum contained thermostable inhibitors of cathepsins B and H, but these molecules had relatively high molecular weights. Therefore the low-molecular-weight 1s and IH from other tissues appear to be intracellular in origin. When extracts of kidney or liver were autolyzed at pH 3 -6, there was a large increase in cathepsin B or H activity with a concomitant decrease in I B and IH. Inhibitor destruction was apparently catalyzed by a proteinase other than cathepsin D or E. Rat kidney, liver, and spleen were rich sources of cathepsins B and H, the kidney yielding three times as much activity per gram as liver or spleen. Hog kidney was rich in cathepsin H, but yielded little or no cathepsin B. IB and 1H were also present in protozoa, tuna fish, chicken and toad. The inhibitors from Tetvahi,menapyriformis inhibited the thiol proteinase from this protozoan.

An endogenous inhibitor of cysteine and serine proteinases from spleen

“…Leucocytes [2,7,9,10], spleen [2,8], liver [4,6], skin [1] and other tissues contain not only sericystatin with an acid pI, but also an isoform with a pI near neutrality [9,10]. It was already known that these protein isoinhibitors inhibit intracellular proteinases of cysteine and serine type.…”

“…According to this property we named it serieystatin, indicating its inhibitory property against serine and cysteine proteinases [10]. Multiple molecular forms exist of this type of intracellular inhibitor [4,9]. Their Mr-values range from 11 000-13 000 and, they differ in pI and intracellular location, and in inhibitory activity against various proteinases.…”

Purification and Characterization of Serine Proteinase Inhibitor from CarpCyprinus carpioOrdinary Muscle

“…Polyacrylamide gel electrophoresis in the presence and absence of SDS was done as described by Weber and Osborn1 5 ) and Davis,16) respectively. Preparative polyacrylamide gel electrophoresis was done by a modification of the analytical method of Davis,16) using an apparatus (Furecovera 11 17 ») from Fuji-Riken Co., Ltd. Five percent and 7.5% polyacrylamide gels (20 x 40mm) were made with 0.75 M Tris-0.25 M HCI buffer, pH 9.0, as a separating gel and a stopper gel, respectively, with 25 mM Tris-0.2 M glycine buffer, pH 8.7, as the electrode buffer, and protein was recovered ·by 12.5mM Tris-O.l M glycine buffer, pH 8.7, with a peristaltic pump at a flow rate of 0.5 ml per min.…”

Some Properties and Postmortem Changes of Cysteine Protease Inhibitors from Rabbit Skeletal Muscles

“…Lenney et a/. 12 ) reported that rat lung cathepsin B inhibitor retained 40% of its activity on a 10-min incubation at 80°C (pH 7.0). Thus, the muscle cathepsin B inhibitors seemed to be less heat-stable than the corresponding inhibitor in rat lung.…”

“…There are many papers describing the purification of cysteine protease inhibitors from various animal tissues including skin,s ""'8,11) liver, 9,10,12,13) spleen, 14) kidney,12) and lung. 11 ,12) Jarvinen 11 ) demonstrated the occurrence of two papain inhibitors showing Mw of 13,400 and 74,000 respectively, in rat skeletal muscles.…”