T-kinin is released from T-kininogen by consecutive cleavage by cathepsin E-like proteinase and 72 kDa proteinase

Using highly purified T-kininogen and cathepsin E-like proteinase and 72 kDa proteinase in rat spleen, the release of T-kinin from T-kininogen was found to occur by consecutive cleavage by cathepsin E-like proteinase and 72 kDa proteinase. 72 kDa proteinase seems to be serine proteinase, because it was completely inhibited by diisopropyl fluorophosphate but not by pepstatin, leupeptin and bestatin.     

Ile-Ser-bradykinin (T-kinin) and Met-Ile-Ser-bradykinin (Met-T-kinin) are released from T-kininogen by an acid proteinase of granulomatous tissues in rats

An acid proteinase of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. The kinin isolated by n-butanol extraction was separated by reverse-phase high-performance liquid chromatography into T-kinin and a T-kinin derivative. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, the T-kinin derivative was identified as Met-Ile-Ser-bradykinin (Met-T-kinin).     

T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.     

Identification of T-kinin-Leu(T-kinin-containing peptide) released from T-kininogen by cathepsin D of granulomatous tissues in rats

Acid proteinases of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. By column chromatography on pepstatin-Sepharose 4B, two types of acid proteinase seems to be responsible for kinin release. One of the acid proteinase was identified as cathepsin D from SDS-polyacrylamide gel electrophoresis and Western-blot analysis, using anti-rat liver cathepsin D IgG. Cathepsin D alone could not release T-kinin, but T-kinin-containing peptides. The T-kinin-containing peptides were separated into two peptides by reverse-phase high-performance liquid chromatography. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, one of the T-kinin-containing peptides was identified as T-kinin-Leu.     

Nature of proteinase inhibitors released from soybeans during imbibition and germination

Proteinase inhibitors are released to a smaller extent from soybeans which have been pre-equilibrated to an atmosphere of high relative humidity (r.h.) compared to those equilibrated to low r.h. Seeds pre-equilibrated to high r.h. also exhibited better germination. Regardless of the states of seed hydration, both Kunitz and Bowman-Birk soybean trypsin inhibitors are released in parallel with respect to each other and to other proteins at germination times up to 100 hr. After 48 hr of germination, new forms of trypsin inhibitor appear in the leachate which react with anti-Bowman-Birk trypsin inhibitor antibodies. No new forms of Kunitz trypsin inhibitor were observed immunochemically during the first 100 hr of germination.     

Selective cleavage of glycyl bonds by papaya proteinase IV

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO₂ is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in β-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.     

C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate

Proteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with alpha-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose-glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS-PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS-PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide-tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Substrate specificity of the serine proteinase from Bacillus subtilis, strain 72

A comparative study of the hydrolysis of various p-nitroanilide substrates (Z-A2-A1-pNA, Z-A3-A2-A1-pNA, and Z-A4-A3-A2-A1-pNA, where A1-An are various amino acid residues, Z is the benzoyloxycarbonylic group and pNA is the p-nitroanilide group), catalyzed by serine proteinase from Bacillus subtilis strain 72, was carried out. It was found that depending on the substrate structure, the hydrolysis may involve both the peptide-p-nitroaniline and the amino acid-amino acid bonds. A kinetic analysis of substrate hydrolysis occurring simultaneously at these two bonds was carried out. The physico-chemical meaning of the kinetic parameters of the given scheme was determined. The quantitative estimation of the enzyme specificity with respect to both hydrolyzing bonds can be found by using the parameters calculated during the analysis of the kinetic curve of p-nitroaniline production. It was found that according to their specificity the amino acid residues at position A1 can be arranged in the following order: L-Leu greater than P-Phe greater than L-Ile greater than L-Ala. The beta-branched amino acid residues, L-Val and L-Ile, do not bind to subsite S1. If these residues occupy position A1, the substrate splitting occurs exclusively between residues A1 and A2. The tetrapeptide N-protected p-nitroanilide substrates are also hydrolyzed at this bond. Partial hydrolysis of the amino acid-amino acid bond between residues A1 and A2 occurs in two cases: i) when residue A1 is loosely bound to subsite S1 and/or, ii) when residue A2 is firmly bound to subsite S1.     

Isolation and characterization of folded fragments released by Staphylococcal aureus proteinase from the non-histone chromosomal protein HMG-1

HMG-1 was isolated from newborn calf thymus without exposure to overt denaturing conditions. The purified protein was digested under several solvent conditions with the proteinase (endoproteinase GluC) from Staphylococcus aureus strain V8. We found that the preferred site of attack by the enzyme on HMG-1 was influenced markedly by ionic strength and temperature. In 0.35 M NaCl/50 mM Tris-phosphate (pH 7.8) at 37 degrees C, cleavage near the junction between the A and B domains is predominant, as previously reported by Carballo et al. (EMBO J. 2 (1983) 1759-1764). However, in 50 mM Tris-phosphate (pH 7.8) lacking NaCl and at 0 degrees C, cleavage between the B and C domains strongly predominates. Three major products of the digestions were purified and characterized. The fragment consisting of domains B and C was found by circular dichroism to contain a substantial amount of helix. This re-emphasizes the importance of avoiding overt denaturing conditions when working with members of the HMG-1 family.     

GLUT4-containing vesicles are released from membranes by phospholipase D cleavage of a GPI anchor

We have previously developed a cell-free assay from rat skeletal muscle that displayed in vitro glucose transporter 4 (GLUT4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT4 transfer assay, we have purified a glycosylphosphatidylinositol (GPI) phospholipase D (PLD) that induces transfer of GLUT4 from small to large membranes. The in vitro GLUT4 transfer was activated and inhibited by suramin and 1,10-phenanthroline (an activator and an inhibitor of GPI-PLD activity, respectively). Furthermore, upon purification of the GLUT4 transporter protein, the protein displayed an elution profile in which the molecular mass was related to the charge, suggesting the presence or absence of phosphate. Second, by photoaffinity labeling of the purified GLUT4 with 3-(trifluoromethyl)-3-(m-[(125)I]iodopenyl)diazirine, both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of Triton X-114, the purified GLUT4 was found to be partly detergent resistant, which is a known characteristic of GPI-linked proteins. Fourth, the purified GLUT4 protein was recognized by an antibody raised specifically against GPI links. In conclusion, GLUT4-containing vesicles may be released from a membrane compartment by action of a GPI-PLD.     

The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component

Endothelial-monocyte-activating polypeptide II (EMAPII) is an inflammatory cytokine released under apoptotic conditions. Its proEMAPII precursor proved to be identical to the auxiliary p43 component of the aminoacyl-tRNA synthetase complex. We show here that the EMAPII domain of p43 is released readily from the complex after in vitro digestion with caspase 7 and is able to induce migration of human mononuclear phagocytes. The N terminus of in vitro-processed EMAPII coincides exactly with that of the mature cytokine isolated from conditioned medium of fibrosarcoma cells. We also show that p43/proEMAPII has a strong tRNA binding capacity (K(D) = 0.2 microm) as compared with its isolated N or C domains (7.5 microm and 40 microm, respectively). The potent general RNA binding capacity ascribed to p43/proEMAPII is lost upon the release of the EMAPII domain. This suggests that after onset of apoptosis, the first consequence of the cleavage of p43 is to limit the availability of tRNA for aminoacyl-tRNA synthetases associated within the complex. Translation arrest is accompanied by the release of the EMAPII cytokine that plays a role in the engulfment of apoptotic cells by attracting phagocytes. As a consequence, p43 compares well with a molecular fuse that triggers the irreversible cell growth/cell death transition induced under apoptotic conditions.     

Serotonin is released from isolated leech ganglia by potassium-induced depolarization.

1. The quantities of serotonin that are released from isolated leech ganglia in vitro were measured with the sensitive neurochemical techniques of HPLC-EC. 2. Segmental ganglia were exposed to elevated concentrations of potassium that depolarize leech serotonin-containing neurons by approximately 35 mV per decade. 3. Each segmental ganglion released on average 0.20 pmol of serotonin during 10 min of incubation in a solution containing 64 mM K+. 4. The rate of serotonin release increased nearly four-fold to 0.74 pmol/10 min when ganglia were incubated in 120 mM K+. 5. The rates of ganglionic serotonin release in 120 mM K+ were quantitatively similar in these three, experimentally important species of leeches: Hirudo medicinalis, Macrobdella decora and Haementeria ghilianii. 6. Ionic substitution experiments with the divalent cations Mg2+ and Co2+ indicated that the release of serotonin from leech ganglia is mediated by a Ca2+ dependent process. 7. The serotonin-uptake blockers, imipramine and chlorimipramine, did not increase the amount of serotonin released in elevated potassium. 8. Vitally staining the identified serotonin-containing neurons with Neutral Red dye did not reduce the quantity of serotonin that was released from the ganglia in elevated potassium. 9. This study demonstrates the capacity of leech ganglia to release the neurochemical serotonin, and the rates of transmitter release increase with the degree of depolarization of serotonin-containing neurons.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria

To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [(3)H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [(3)H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria.     

The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase.

The 2A proteinases (2Apro) of certain picornaviruses induce the cleavage of the eIF4G subunit of the cap-binding protein complex, eIF4F. Several reports have demonstrated that 2Apro of rhinovirus and coxsackievirus B4 cleave eIF4G directly. However, it was suggested that in poliovirus infection, the 2Apro induces the activation of a cellular proteinase which in turn cleaves eIF4G. Furthermore, it is not clear whether eIF4G is cleaved as part of the eIF4F complex or as an individual polypeptide. To address these issues, recombinant eIF4G was purified from Sf9 insect cells and tested for cleavage by purified rhinovirus 2Apro. Here we report that eIF4G alone is a relatively poor substrate for cleavage by the rhinovirus 2Apro. However, an eIF4G-eIF4E complex is cleaved efficiently by the 2Apro, suggesting that eIF4F is a preferred substrate for cleavage by rhinovirus 2Apro. Furthermore, 2Apr drastically reduced the translation of a capped mRNA. An eIF4G-eIF4E complex, but not eIF4G alone, was required to restore translation.     

Initial site of cleavage towards oxidized B-chain of insulin by thermophilicStreptomyces alkaline proteinase

The initial site of cleavage towards the oxidized B-chain of insulin by thermophilicStreptomyces alkaline proteinase (EC 3.4.21.14) was investigated. The most susceptible bond was between Leu¹⁵ and Tyr¹⁶. Additional cleavage of the bonds Leu¹¹-Val¹², Leu¹⁷-Val¹⁸, and Gln⁴-His⁵ were noted. The results suggested that the specificity of bond, cleavage of thermophilicStreptomyces alkaline proteinase is close to that of subtilisin Carlsberg.