Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.     

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.     

Purification and Characterization of Cynomolgus Monkey Tryptase

Cynomolgus monkey tryptase was purified to homogeneity from lung tissue. Reducing SDS-PAGE analysis of the monkey enzyme produced a doublet at 30–32 kDa, which reacted with antibodies against human lung tryptase on a Western blot. N-terminal sequence analysis of the monkey enzyme yielded a sequence that was identical to human tryptase out to 15 residues. Gel filtration chromatography either in the presence or absence of heparin indicated that the monkey enzyme had a molecular mass of approximately 250 kDa and 140 kDa, respectively, consistent with the formation of a tetramer that can bind heparin. Other similarities between human and monkey tryptase included the ability to degrade vasoactive intestinal peptide and a resistance to inhibition by biological serine protease inhibitors. However, the two enzymes displayed markedly different pH stability profiles. Monkey tryptase was unstable at pH values over 7.0, even in the presence of heparin, displaying a half-life of 10.9 min at pH 8.0. In addition, the stabilizing effect of heparin was pH dependent, being most prevalent at lower pH values. Therefore, the biological activity of monkey tryptase may be controlled by both pH and the availability of heparin.     

Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.     

Characterization of human, bovine, and horse antithrombin III.

A comparison of the physical-chemical properties of human, bovine, and horse antithrombin III has been made. These three plasma proteins are strong inhibitors of bovine factor Xa and form a 1:1 molar complex with this coagulation enzyme. Human, bovine, and horse antithrombin III are glycoproteins containing hexose, hexosamine, and neuraminic acid. The total carbohydrate was 9, 12, and 16% for human, bovine, and horse antithrombin III, respectively. These proteins have a similar amino acid composition, although some monor variations were noted. Each antithrombin III is composed of a single polypeptide chain with an amino-terminal histidine residue. Of the first 17 amino-terminal residues, only three differences were noted between the three proteins. These occur in position 2 which is occupied by Gly, Arg, and Trp in human, bovine, and horse, respectively; position 6 which has a deletion in human antithrombin III; and position 8 where Ile in human and horse antithrombin III has been replaced by Val in the bovine preparation. The remainder of the first 17 residues is the same in all three proteins. The molecular weights for the bovine and horse preparation were 56 600 and 52 500, respectively, as determined by sedimentation equilibrium in the presence of guanidine hydrochloride. Some immunological cross-reactivity was also observed between the three different proteins.     

Interactions of human mast cell tryptase with biological protease inhibitors.

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.     

Human histidine-rich glycoprotein: simultaneous purification with antithrombin III and characterization of its gross structure

The simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22 mg and 70 mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50,000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30,000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region(s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W.T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an amino-terminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.     

Bovine tryptases. cDNA cloning, tissue specific expression and characterization of the lung isoform.

A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26-amino acid prepro-sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp-Ser are present at positions 188-189, while in BLCT these positions are occupied by residues Asn-Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in-gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35-40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (kcat/Km) assayed with model substrates are 10- to 60-fold higher in the case of BLT. The tissue-specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.     

Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae

Yeast alpha-mannosidase, a marker enzyme of vacuolar membranes, was solubilized and purified from commercial bakers' yeast. The alpha-mannosidase was solubilized efficiently with 10 mM Na2CO3. A high pH (greater than 8.5) and a sufficient amount of a detergent such as 0.2% (w/v) Triton X-100 were required to keep the enzyme in a soluble state. This suggested that the enzyme is either a peripheral membrane protein or an ecto-type integral membrane protein. After 4,300-fold purification by conventional chromatography, the alpha-mannosidase gave a single band on nondenaturing polyacrylamide gel electrophoresis, but could be fractionated into active isoforms, which consisted of 107-, 73-, and 31-kDa polypeptides, with a Mono Q anion exchange fast protein liquid chromatography system. Apparent molecular weight of the native enzyme was determined as 560,000. It suggested that the composition of isoforms will be described as (107 kDa)n (73 kDa)6-n (31 kDa)6-n, where n is 0-6. The 107- and 73-kDa polypeptides were purified further under denaturing conditions. One-dimensional peptide map analysis and immunological analysis of these polypeptides indicated that they are closely related proteins. Immunoblotting of crude cell lysates revealed that the 107-kDa polypeptide appeared first, and then the 73-kDa polypeptide appeared along growth phase. It suggested that proteolytic conversion of the 107-kDa polypeptide occurs to form the 73- and 31-kDa polypeptides and leads to formation of isoforms of the enzyme.     

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

Probing the heparin-binding domain of human antithrombin III with V8 protease

From structural analysis on genetically abnormal and chemically modified human antithrombin III [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81, 289-293; Chang, J.-Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174-1176; Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941], the heparin-binding site of antithrombin III has been suggested to be in the region of Pro-41, Arg-47 and Trp-49. In this study the heparin-binding site was probed by preferential cleavage of V8 protease on heparin-treated and non-treated native antithrombin III. The study has been based on the presumption that the heparin-binding site of antithrombin III is situated at exposed surface domain and may be preferentially attacked during limited proteolytic digestion. Partially digested antithrombin III samples were monitored by quantitative amino-terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1-h-digested antithrombin III was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu-Gly (residues 34-35), Glu-Ala (residues 42-43) and Glu-Leu (residues 50-51) are three preferential cleavage sites for V8 protease and their cleavage, especially the Glu-Ala and the Glu-Leu sites, was drastically inhibited when antithrombin III was preincubated with heparin. Both high-affinity and low-affinity antithrombin-III-binding heparins were shown to inhibit the V8 protease digestion of native antithrombin III, but the high-affinity sample exhibited a higher inhibition activity than the low-affinity heparin. These findings (a) imply that the segment containing residues 34-51 is among the most exposed region of native antithrombin III and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.     

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for No Proteolytic Processing during Transport of Isocitrate Lyase into Glyoxysomes in Castor Bean Endosperm

The amino-terminal sequence of isocitrate lyase purified fromcastor bean endosperm glyoxysomes was compared with that deducedfrom the nucleotide sequence of cDNA for the enzyme [Plant Mol.Biol. (1987) 8: 471]. The isolated active enzyme lacked sixamino acid residues in the amino terminus, although the enzymeimmunoselected from a tissue homogenate with trichloroaceticacid had the amino-terminal part. Thus the six amino acid residuesseem to be eliminated during enzyme purification and the enzymeis transported into glyoxysomes without proteolytic processing. (Received August 31, 1987; Accepted November 30, 1987)     

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.     

The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH

The novel tetrameric structure of human beta-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human beta-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-beta-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to beta-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-beta-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 beta-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts beta-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.     

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.     

Screening and characterization of trehalose-oleate hydrolyzing lipase

Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE. One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity. The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X. The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1). The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme. The pI of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C. Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3). High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane. The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species. Thus the enzyme was concluded to be a kind of lipase.