Exoprotease Activity of Two Marine Bacteria during Starvation

Exoprotease activity during 120 h of total energy and nutrient starvation was examined in two marine bacteria, Vibrio sp. strain S14 and Pseudomonas sp. strain S9. The activity was determined by spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder (hide powder azure). Starved cells of both strains (5 h for S14, and 4 or 24 h for S9) showed greater extracellular proteolytic activity than at the onset of starvation. The exoprotease activity of cells starved for longer periods of time then decreased, but was found to be present at significant levels throughout the starvation period studied (120 h). The accumulation of exoprotease activity in the bulk phase during starvation indicated that both strains constitutively excreted extracellular proteases. As deduced from experiments with chloramphenicol, de novo protein synthesis during starvation was required for the production and/or release of the exoproteases into the surrounding environment. The degradation of hide powder azure allowed an immediate increase in respiration rate, also by long-term-starved cells. This suggests that metabolic systems are primed to respond to the availability of substrates, allowing the cells to recover rapidly. The regulation of exoprotease activity was also studied and found to be different in the two strains. Casamino Acids repressed exoprotease activity in Pseudomonas sp. strain S9, whereas a mechanism similar to catabolite repression was found for Vibrio sp. strain S14 in that glucose repressed activity and cyclic AMP reversed this effect. The exoproteases appeared to be metalloproteinases because the addition of EDTA to cell-free starvation supernatants from both strains significantly inhibited the activity of the proteases.     

Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.     

Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol.

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.     

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.     

Characterization and purification of extracellular proteases of Trichomonas vaginalis

Extracellular protease activity was detected in serum-free culture filtrates of Trichomonas vaginalis. The activity was demonstrated by hydrolysis of hide powder azure and possessed the characteristics of cysteine type proteases: inhibition by N-ethyl maleimide, Cu2+, antipain, N-tosyl-L-phenylalanine chloromethyl ketone, N-tosyl-L-lysine chloromethyl ketone, leupeptin, chymostatin, and iodoacetamide, and enhancement by cysteine, EDTA, and dithiothreitol. The activity was optimal at acid pH and the protease was also active on peptide nitroanilides with arginine derivatives. Purification of this activity by ethanol precipitation, ammonium sulfate fractionation, ion exchange chromatography, and gel filtration resulted in the isolation of two proteases estimated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to have molecular masses of 60 and 30 kilodaltons (kDa), respectively. The larger molecular mass protease broke down during purifications to two subunits of approximately 23 and 43 kDa, as determined by gel electrophoresis. Rabbit sera derived by immunization with the 23-kDa subunit cross-reacted by immunoblot with the 60- and 43-kDa subunits, but not with the 30-kDa protease. These soluble products of T. vaginalis growth could be important pathogenically in establishing T. vaginalis infection in the normally acid (pH less than or equal to 4.5) environment of the vagina.     

Proteolytic activities of cobra venoms based on inactivation of alpha 2-macroglobulin

The venoms of various cobra species showed a wide range of abilities to cleave hide powder azure, with Naja naja kaouthia and Ophiophagus hannah venoms showing the lowest activities and Naja nivea venom showing the greatest activity on this dye-linked substrate. The activities of the venoms on hide powder did not completely correlate with their ability to inactivate the alpha 2-macroglobulin of human serum. Incubation of 4-5 micrograms of Naja nigricollis venom per microliter of serum for 30 min caused loss of 95% of the alpha 2-macroglobulin activity of the serum. The inactivation was rapid, reaching 80% inactivation 5 min after mixing. This loss of alpha 2-macroglobulin activity was used to quantitate the weak proteolytic activity of N. nigricollis venom and a partially purified sample of the major fibrinogenolytic proteinase of the venom. The inactivation of alpha 2-macroglobulin was also used to compare the proteinase activities of venoms from seven species or subspecies of cobra. Based on alpha 2-macroglobulin inactivation, N. nigricollis had the highest proteinase activity among the tested venoms. The measurement of alpha 2-macroglobulin inactivation should provide a useful alternative to hide powder digestion for demonstration of weak proteolytic activities in venoms.     

Effect of alpha2M on earthworm fibrinolytic enzyme III-1 from Lumbricus rubellus

Though it is known that human alpha2-macroglobulin (alpha2M) inhibits most proteases, the effect of alpha2M has not been investigated on earthworm fibrinolytic enzymes (EFEs) from Lumbricus rubellus, which could be transported from intestine epithelium into blood as an intact molecule (Fan et al., Biochim. Biophys. Acta 1526 (2001) 286). The activity of earthworm fibrinolytic III-1 (EFE-III-1) decreased to 65% when incubated with alpha2M, while it decreased to 30% in plasma under the same conditions. The first order rate of the inactivation of EFE-III-1 with alpha2M was similar to that of fast phase with plasma, indicating that alpha2M may be the inhibitor initially binding to the enzyme in blood. SDS-PAGE showed that incubation of EFE-III-1 with alpha2M a released fragment ( approximately 90 kDa), followed by formation of a high molecular weight complex (approximately 700 kDa). There was a linear relationship between the apparent inhibition rate constant (k1) and [alpha2M], by double reciprocal plot. It was suggested, as described by Tsou (Acta Biochem. Biophys. Sinica 5 (1965) 398) and Tian (Biochem. J. 21 (1982) 1028), that the mechanism of alpha2M/EFE-III-1 interaction could be coincided with a complexing irreversible inhibition. Experiments in both the inactivation and the intrinsic fluorescence showed that alpha2M bound to the enzyme mole by mole equivalently. The intrinsic fluorescence of alpha2M was enhanced with an observable blue shift in emission maxima, suggesting that alpha2M was one of the important inhibitors to EFEs when it absorbed into blood.     

Isolation, purification, and properties of Penicillium charlesii alkaline protease.

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.     

The kinetics of inhibition of alpha-thrombin in human plasma

Methods have been developed for kinetic studies of the inhibition of exogenous unmodified thrombin in human plasma containing normal levels of fibrinogen and calcium ion. To prevent interference by other proteases, factor VIII-deficient plasma was used and contained 50 nM Phe-Phe-Arg-chloromethyl ketone and 1 kallikrein-inactivating unit/ml aprotinin; neither inhibited thrombin at these levels. Two independent assays were used. The first was the discontinuous amidolytic assay of thrombin activity, which measures both free thrombin and thrombin-alpha 2-macroglobulin complex, and was used to estimate the rates of inhibition both by "inactivating" inhibitors, such as anti-thrombin and alpha 1-protease inhibitor, and by alpha 2-macroglobulin (alpha 2M). The contribution of alpha 2M was confirmed by a second method, which measured with time the generation of amidolytic activity due to the thrombin-alpha 2M complex. The total rate of thrombin inhibition in plasma containing 4 mM free Ca2+ was of the order of 1.9 min-1, of which 0.4 min-1 was due to alpha 2M and 0.9 min-1 was due to inhibitors that were removed when plasma was passed through heparin-agarose. Thrombin inhibition was also measured in varying dilutions of plasma and confirmed that total inhibition rate is approximately linearly related to plasma (and thus inhibitor) concentration. Negatively charged phospholipid micelles had very little effect on thrombin inhibition rate, but platelets accelerated inhibition to more than 2.5 min-1.     

Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.     

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.     

Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme.

An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa.     

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei)

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.     

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.     

Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.     

Purification and Characterization of Beauveria bassiana Proteinases

Two chymotrypsin‐like serine proteinases are produced by B. bassiana 278 when grown on different carbon and nitrogen sources. By employing acetone precipitation, gel filtration and ion‐exchange chromatographies, the enzymes were separated from the culture filtrate after propagation of the fungus on medium enriched either with ground larvae of Apis mellifera (Proteinase I) or porcine blood plasma (Proteinase II). The purified enzymes with a molecular mass of approximately 32 kDa hydrolyzed natural protein substrates: casein, hide powder azure (HPA), azocoll and much less elastin Congo Red and collagen. They differ from each other in the optimum pH value, amino acid composition, Michaelis constant and susceptibility to natural chymotrypsin inhibitors. Both proteinases hydrolyze suc‐Ala‐Ala‐Pro‐Phe‐p‐NA with an apparent K_m of 2.03 × 10^—3 M and 1.04 × 10^—4 M, respectively. The turkey ovomukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Proteinase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be very high (4.11 × 10⁹ M^—1).     

Purification and characterization of alpha2-macroglobulin from grass carp Ctenopharyngodon idellus: cloning a segment of the corresponding gene

The plasma protein alpha2-macroglobulin (alpha2M) was purified by gel filtration and anion-exchange chromatography from grass carp plasma. The alpha2M consists of two different subunits of molecular weight 95 kDa and 80 kDa, respectively. The characteristics of grass carp alpha2M are similar to mammalian alpha2M, in that grass carp alpha2M exists in two forms: a fast-form and a slow-form. The former is complexed with protease. The sequence of grass carp alpha2M-conserved region and a region containing the bait region was determined by sequence analysis using polymerase chain reaction (PCR). The deduced amino acid sequence of the conserved region is similar to the alpha2M sequence of common carp, however, the bait region amino acid sequence is dramatically distinct from that of common carp. This may partially explain the differential ability of alpha2M of different species to inhibit different proteases. The alpha2M was able to inhibit Aeromonas hydrophila extracellular protease (AhECPase) and thus may play a role in resistance to infection by this pathogen.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.