A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.     

The isolation and characterization of a new elastase inhibitor,pre-α2-elastase inhibitor,of the horse

A new and probably unique elastase inhibitor of horse serum was identified, purified to homogeneity and called pre-α₂-elastase inhibitor of the horse. Electrophoretically it migrated immediately in front of the α₂ position. Its molecular weight was 188 000 by pore limit polyacrylamide gel electrophoresis and 225 000 by Sephadex G-200 gel filtration. The inhibitor was composed of at least two non-identical polypeptide chains of M_r 68 400 and 87 600. A banding pattern of restricted heterogeneity focused between pH 4.9 and 5.2 was revealed by isoelectric focusing. Of 13 animal, microbial and plant proteinases, horse pre-α₂-elastase inhibitor inhibited only pancreatic elastase and trypsin efficiently. Chymotrypsin was inhibited only in traces. No analogy between the elastase inhibitor and the known human serum inhibitors could be found with respect to immunological and biochemical criteria.     

Evolution of the proteinase inhibitor I family and apparent lack of hypervariability in the proteinase contact loop

A protein phylogenetic tree was constructed from 24 homologous proteinase inhibitor I sequences identified in the EMBUGenbank and Swiss-Prot databases and from translated amino acid data from four constitutive cDNA clones of proteinase inhibitor I characterized from potato tuber mRNA. The tree suggests that divergence of at least four paralogous proteins with functional specialization occurred at different times during the evolutionary history of the proteinase inhibitor I family. Five distinct regions in the primary structure, earlier identified by structural studies, were used to analyze the inhibitor family for hypervariability (Creighton and Darby, Trends Biochem Sci 14:319–324, 1989). Mutations did not occur with higher-than-random frequency within the proteinase binding region. When isoinhibitor, orthologous, or paralogous data subsets were subsequently analyzed the same results were obtained. Comparison of the amino acid sequences for all the known potato proteinase isoinhibitor I proteins identified ten highly variable sites. These also were distributed randomly. Thus hypervariability, which has been observed in all other serine proteinase inhibitor families to date, appears to be lacking in the proteinase inhibitor I family.     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.     

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-IΔD86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed. Received: 3 July 1997 / Accepted: 8 August 1997     

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300–1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35 000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60°C) and resistant to inactivation by trypsin (2 h, 37°C, 10 μg/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.     

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Summary Previous studies with rats have shown that a single oral dose of the proteinase inhibitor Camostate (FOY-305) induces release of cholecystokinin (CCK) into the circulation, which lasts for 3 to 6h. This transient endogenous release of hormone results in a depletion of pancreatic enzyme stores within 1 h and an increase in total rate of protein synthesis, which peaks at 6 to 9 h. At the level of individual enzyme biosynthesis a transient decrease in amylase and an increase in trypsinogen and chymotrypsinogen is observed. In the present study the time course of DNA synthesis and the labeling index of 5 populations of pancreatic cells have been analysed following a single oral dose of 50 or 100 mg/kg proteinase inhibitor, using in vivo labeling with 12 Ci/g body weight ³H-thymidine 1 h prior to sacrifice of the animals. DNA synthesis did not change during the initial 12 h following inhibitor feeding and then showed a phasic increase with a peak (20-fold) at 24h and intermediate increases (4- to 5-fold) at 18 and 36 h, respectively. From the 5 pancreatic cell populations studied by autoradiography the labeling indices of interlobular duct cells and islet cells did not change over the entire observation period. Acinar cells, intralobular duct cells and interstitial cells showed a marked increase in labeling index with peak values at 24h, which were 20-fold in acinar cells and 5.5- and 8.5-fold in intralobular duct cells and interstitial cells, respectively. The data demonstrate a significant growth response of pancreatic acinar tissue after a single episode of endogenous CCK-release, which is similar in extent, time course and cellular source as previously demonstrated during persistent stimulation of the pancreas by prolonged infusion of the CCK-analogue caerulein.     

Both action potentials and variation potentials induce proteinase inhibitor gene expression in tomato

Tomato plants (Lycopersicon esculentum) accumulate proteinase inhibitor 2 (pin2) mRNA in response to insect attack, crushing and flaming in leaves distant from those treated. Most earlier work suggests that the systemic wound signals are chemical; here we try to determine whether electrical or physical (hydraulic) signals can also evoke pin expression. We used a mild flame to evoke a systemic hydraulic signal and its local electrical aftermath, the variation potential (VP), and we used an electric stimulus to evoke a systemic electrical signal, the action potential (AP). We determined the kinetic parameters of both the VP and AP. Flame-wounded plants essentially always exhibited major electrical responses throughout the plant and a several-fold increase in pin2 mRNA within 1 h. Electrically stimulated plants that generated and transmitted a signal (AP) into the analyzed leaf exhibited similarly large, rapid increases in pin2 mRNA levels. Plants which generated no signal, or signals of just a few microvolts, had unchanged levels of pin2 mRNA. Since the AP and VP both arrived in the receiving leaf before accumulation of pin2 mRNA began, we conclude that, in addition to the previously shown chemical signals, both hydraulically induced VPs and electrically induced APs are capable of evoking pin2 gene expression.     

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine α-chymotrypsin (α-chymotrypsin) [native and with the Met192 residue converted to the sufoxide derivative (Met(O) α-chymotrypsin)] as well as to native bovine β-trypsin (β-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0°C ad 30.0°C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methytonyl residues present at the proteinase: inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation methionyl residues of SLPI and α-chymotrypsin, by the reaction with chloramines T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion native SLPI and native α-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Summary Application of a single dose of a new type of proteinase inhibitor camostate (FOY-305) via orogastric tube was used in rats to study the dose-response relationship of resulting pancreatic stimulation. Doses up to 10 mg/ kg failed to elicit any response, while significant decrease in enzyme content and increase in serum CCK-levels were observed with doses ranging from 25 to 400 mg/kg. A single dose of 100 mg/kg was selected for a time-sequence analysis, which revealed a 60 to 70% depletion of enzyme stores persisting over 6 h and reverting to control levels by 12 h. Peak increases in serum CCK-levels (15-fold above the elevation observed after regular food intake) were found after 30 min and persisted as an 8-to 10-fold elevation for at least 3 h, then declined to control levels by 9 h. This prolonged endogenous hormone release and resulting pancreatic stimulation were also verified in a separate group of animals in which volume, protein, and enzyme output were measured after cannulation of the pancreatic duct. While volume secretion was not altered by feeding a single dose of 100 mg/kg FOY-305, protein and enzyme output increased 2-to 3-fold over a period of 7 h. Fine-structural analysis of the pancreas demonstrated efficient depletion of zymogen granules from acinar cells with all doses between 50 and 400 mg/kg, accompanied by the appearance of membrane material in the acinar lumina at 3 and 6 h. The same transient increase in the number of lysosomal bodies predominantly containing mitochondria with all doses above 50 mg/kg was interpreted as increased organelle turnover due to persisting hormonal stimulation.Supported by a grant from the Deutsche Forschungsgemeinschaft (Ke 113/15-1/2)     

Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression

Summary Two cDNA clones containing the complete coding region of a developmentally controlled (tuber-specific) as well as environmentally inducible (wound-inducible) gene from potato (Solanum tuberosum) have been sequenced. The open reading frame codes for 154 amino acids. Its sequence is highly homologous to the proteinase inhibitor II from tomato, indicating that the cDNA's encode the corresponding proteinase inhibitor II of potato. In addition the putative potato proteinase inhibitor II contains a sequence which is completely homologous with that of another small peptide proteinase inhibitor from potato, called PCI-I. Evidence is presented that this small peptide is probably derived from the proteinase inhibitor II by posttranslational processing.Northern type experiments using RNA from wounded and nonwounded leaves demonstrate that RNA homologous to the putative proteinase inhibitor II cDNA's accumulates in leaves as a consequence of wounding, whereas normally the expression of this gene is under strict developmental control, since it is detected only in tubers of potato (Rosahl et al. 1986). In addition the induction of this gene in leaves can also be achieved by the addition of different polysaccharides such as poly galacturonic acid or chitosan. In contrast to the induction of its expression by wounding in leaves, wounding of tubers results in a disappearance of the proteinase II inhibitor m-RNA from these organs.     

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation.Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective.Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway.The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance.The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.     

Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo)

A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of ∼8 kDa correlating to the peak 8041.5 Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K_i of 309.8 nM) and chymotrypsin (K_i of 10.7 μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 °C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 °C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.     

Solid-phase synthesis and dimerization of an azobenzene-containing peptide as photoisomerizable proteinase inhibitor

Summary The synthesis and biological activity of a photochromic compound, in which two Lys₂ dipeptides are linked through their N-terminal amino groups by an azobenzene moiety, are reported. The synthesis was achieved by a novel solid-phase dimerization strategy, based on the reaction of 4,4-azobenzene dibenzoyl chloride with two N-terminal amino groups of distinct Lys₂ dipeptides directly on the resin. Both the trans form and the photostationary state mixture (59% cis and 41% trans) inhibit a plant proteinase which is known to be sensitive to polycationic compounds.     

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire)

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1 h to 120 °C with a pressure of 1 kg/cm². Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.