Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

Comparison of muscle proteinase activity among fish species

The effects of temperature during storage, portion of muscle and growth stage of fish on the activity level of carp muscle acid (cathepsin D, EC 3.4.23.5), neutral and alkaline proteinases were examined. Icing storage, freezer storage and portion of muscle did not affect each proteinase activity (P less than 0.05), but acid proteinase activity was affected by growth stage (P less than 0.05) and the level decreased from small to large fish. The activity levels of three kinds of proteinases were compared among species (P less than 0.05) and the following order was obtained. Acid proteinase, white croaker = rainbow trout = carp greater than sardine = common mackerel, sardine greater than lizardfish, common mackerel = lizardfish. Neutral proteinase, rainbow trout greater than carp = white croaker greater than lizardfish = sardine = common mackerel. Alkaline proteinase, rainbow trout = sardine greater than white croaker = carp = common mackerel greater than lizardfish.     

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.     

Inhibition of in-vitro fertilization of hamster oocytes by a thiol proteinase inhibitor

Amongst the proteinase inhibitors tested, thiolstatin, a specific inhibitor for the thiol proteinases, leupeptin and antipain, both specific inhibitors of serine- and thiol-proteinases, strongly reduced fertilization of hamster oocytes in vitro. These results suggest the possible involvement of thiol proteinase(s), as well as acrosin, in the fertilization process. A possible role for thiol proteinase in sperm adhesion to the zona pellucida is proposed.     

Amino acids and peptides. XV. Synthesis of Gln-Val-Val-Ala-Gly and derivatives, a common sequence of thiol proteinase inhibitors and their effects on thiol proteinase

The Gln-Val-Val-Ala-Gly sequence, which occurs frequently in several natural thiol proteinase inhibitors, and derivatives were synthesized by conventional solution methods and their effect on thiol proteinases were examined. The studies led us to the conclusion that certain of these peptides exhibited a weak inhibitory effect on the thiol proteinase, papain. One of them, Z-Gln-Val-Val-Ala-Gly-OMe, showed a protective effect on papain from natural thiol proteinase inhibitor-induced inactivation. The relationship between structure and activity of these derivatives was studied and certain conclusions were derived on possible mode of action of these inhibitors. From these studies, it was concluded that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit some effect on papain.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Acidic thiol proteinase activity of Schistosoma mansoni egg extracts.

Extracts of the eggs of the human blood fluke, Schistosoma mansoni, exhibit proteolytic activity which requires the presence of added thiol reagents or cyanide. The pH optimum for hydrolysis of Azocoll and cartilage proteoglycan is 4.8--5.2 and the molecular weight of the major component is 25--26,000. The effects of inhibitors suggest this activity belongs to the acidic thiol proteinase class, with a similarity to Cathepsin B. These proteinases may be involved in nutrition of the egg or sporocyst, in penetration of eggs or miracidia through host tissues, or in the immunopathology of schistosomiasis.     

Effect of various proteinase inhibitors on ovulation of explanted hamster ovaries

Hamster ovaries explanted at 21:00-24:00 h on the day of pro-oestrus were incubated with microbial proteinase inhibitors until 10:30 h on the next morning and the ovulatory blocking effect of these inhibitors was examined. Amongst 11 proteinase inhibitors examined, talopeptin, a specific inhibitor for metallo-proteinases, and alpha-MAPI, a specific inhibitor for serine and thiol proteinases, were the strongest blockers. These 2 inhibitors exhibited a chronological discrepancy in their blocking effect on ovulation. S-SI, plasminostreptin, elastatinal, antipain and chymostatin, which are inhibitors for serine proteinases, partly but significantly inhibited ovulation. The results suggest that, in addition to a metallo-proteinase reported previously, a proteinase which is sensitive to alpha-MAPI is essential for the ovulatory process, and that serine proteinase(s) also participate in ovulation of the hamster ovary.     

Alkaline proteinase localization in myoblasts

Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.     

Autolysis in the tail muscles of metamorphosing tadpoles

Degradation of muscle homogenate from the metamorphosing tadpole tail of bullfrog, Rana catesbeiana, was examined at acid and neutral pHs. More rapid and complete degradation was observed at acid pH. Proteinases working at acid pH were not inhibited by pepstatin but were inhibited by leupeptin. However, the inhibition by leupeptin was enhanced by pepstatin. These results show that lysosomal proteinases, a thiol proteinase(s) rather than cathepsin D, are involved in the degradation of tail muscle proteins.     

High molecular weight heat-stable alkaline proteinase from white croaker and chum salmon muscle: comparison of the activating effects by heating and urea

Effects of heating and urea on the heat-stable alkaline proteinase from white croaker and chum salmon muscle were compared in order to know the regulating mechanism of the proteinase. Chum salmon proteinase required a higher temperature for activity and was more heat-stable than white croaker proteinase. In the presence of 5M urea, the activity was observed to some degree at 37 degrees C only in white croaker proteinase, while both proteinases lost their activities at usual assay temperature around 60 degrees C. These results suggest that the stability of the regulatory and catalytic subunits of the proteinases is somewhat different among fish species.     

The multicatalytic proteinase of mammalian cells

A high-molecular-weight nonlysosomal proteinase has recently been discovered in mammalian cells. It is a widely distributed and abundant enzyme which has attracted attention because of its complex multisubunit structure and its unusual catalytic properties. The 700-kDa proteinase is composed of many different types of low-molecular-weight subunits (Mr 21,000-34,000) arranged in a hollow cylindrical structure. This 20 S complex is very similar, if not identical, to the 19-20 S cylindrical particles, ring-type particles, or prosomes which have been isolated from several different types of eukaryotic cells. The proteinase appears to have at least two distinct catalytic sites and can cleave bonds on the carboxyl side of basic, hydrophobic, or acidic amino acid residues. Inhibition of proteinase activity by thiol reagents supports the suggestion that the enzyme is a cysteine proteinase but there is some evidence that it may be a serine proteinase and the catalytic mechanism is at present unknown. ATP has little effect on proteinase activity in most purified preparations but recently the proteinase has been implicated in ATP-dependent pathways of protein degradation. Ther is a second type of high-molecular-weight complex multisubunit proteinase, a 26 S particle, which catalyzes the ATP-dependent degradation of ubiquitin-protein conjugates. The precise function of these two complex proteinases in intracellular proteolysis remains to be elucidated.     

Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade

Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.     

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactivate up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145 000 to 172 000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.     

Parathyroid hormone increases thiol proteinase activity by activation of protein kinase C in cultured kidney tubule cells (OK)

Chronic exposure (24 h) to parathyroid hormone (PTH) increases the intracellular proteolytic activity in cultured opossum kidney cells 2-fold at physiological PTH concentrations (10(-12) mol/l). This increase can be blocked by E-64, an inhibitor of thiol proteinases. The phorbol ester TPA mimicks the effect of PTH, whereas the calcium ionophore A23187 reduces the intracellular proteinase activity. Forskolin and dibutyrylic cAMP do not elevate proteinase activity. The protein kinase C inhibitor staurosporine is equally effective in blocking the TPA- and PTH-induced proteinase activity increase. These data indicate that PTH increases the intracellular thiol proteinase activity by an activation of protein kinase C and not by the cAMP dependent way.     

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.     

Thiol-dependent serine proteinase from Streptomyces thermovulgaris

The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.