Apparent Subunit Molecular Weight Research Articles

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001.

A nitroreductase-encoded gene from an efficient nitro-reducing bacterium Streptomyces mirabilis DUT001, named snr, was cloned and heterogeneously expressed in Escherichia coli. The purified Streptomyces nitroreductase SNR was a homodimer with an apparent subunit molecular weight of 24kDa and preferred NADH to NADPH as a cofactor. By enzyme incubation and isothermal calorimetry experiments, flavin mononucleotide (FMN) was found to be the preferred flavin cofactor; the binding process was exothermic and primarily enthalpy driven. The enzyme can reduce multiple nitro compounds and flavins, including antibacterial drug nitrofurazone, priority pollutants 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates 3-nitrophthalimide, 4-nitrophthalimide, and 4-nitro-1,8-naphthalic anhydride. Among the substrates tested, the highest activity of kcat(app) /Km(app) (0.234μM-1 Sec-1 ) was observed for the reduction of FMN. Multiple sequence alignment revealed that the high FMN reduction activity of SNR may be due to the absence of a helix, constituting the entrance to the substrate pocket in other nitroreductases.

Cloning, expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from Thermoanaerobacter tengcongensis.

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60kDa. Kinetics assay indicated that TtG6PD preferred NADP(+) (k cat/K m=2618mM(-1)s(-1), k cat=249s(-1), K m=0.10±0.01mM) as cofactor, although NAD(+) (k cat/K m=138mM(-1)s(-1), k cat=604s(-1), K m=4.37±0.56mM) could also be accepted. The K m values of glucose-6-phosphate were 0.27±0.07mM and 5.08±0.68mM with NADP(+) and NAD(+) as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8-9.0 for NADP(+) and at pH 7.0-8.6 for NAD(+) while the optimal temperature was 80°C for NADP(+) and 70°C for NAD(+). This was the first observation that the NADP(+)-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD(+)-linked and growth (75°C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.

Expression of Functional Region of Peanut (Arachis hypogaea) Agglutinin Gene as an Active Lectin in Insect Cells

Natural peanut agglutinin (PNA) gene is expressed with a signal sequence of 23 amino acids and a C terminal peptide of 14 amino acids. Functionally active recombinant PNA having apparent subunit molecular weight of 29kD was obtained when expressed without signal peptide and non-essential C terminal peptide sequences in insect cells. Expression in insect cells (Sf9) was driven by a 129bp Spodoptera litura nucleopolyhedrosis virus (S/NPV) sequence containing its polyhedrin promoter.

Purification and Cloning of TF2: A Novel Protein That Binds a Regulatory Site of the gp2 Promoter in Dictyostelium

The glycogen phosphorylase-2 gene is developmentally regulated and plays a central role in cellular differentiation in Dictyostelium. There are two isozymes of glycogen phosphorylase, GP1 and GP2. Both forms are developmentally regulated; gp1 is expressed in undifferentiated cells and gp2 during differentiation. We report here the identification, purification, and cloning of a second gp2 DNA-binding factor called TF2. This protein demonstrates a high specificity for a transcriptional regulatory element, the 5′ C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5′ C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS–PAGE. SDS–PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. Southern blot analysis indicated that there is only one form of the tf2 gene. Northern analysis showed little or no expression of tf2 in undifferentiated cells. During development tf2 expression increases up to a maximum at 8 h and then decreases in later stages. Attempts to disrupt the gene suggest that tf2 mutation may be lethal.

PURIFICATION AND PARTIAL CHARACTERIZATION OF TYROSINASE ISOFORMS FROM CAP FLESH OF PORTABELLA MUSHROOMS

Tyrosinase was isolated from the cap flesh of Portabella mushrooms. Several mg of enzyme were isolated with an overall yield of 18%. Large DEAE ion exchange columns and selective pooling of fractions, followed by chromatography of pooled fractions on hydroxylapatite columns resulted in the isolation of highly purified tyrosinase. The isolated enzymes had apparent subunit molecular weights of approximately 48–50 kDa and pIs from 5.1 to 5.3. The purification scheme can be carried out rapidly and has the potential to separate different groups of tyrosinase isoforms.

Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization

To better understand the genetic disorder Sjögren–Larsson syndrome which is caused by a deficiency of fatty aldehyde dehydrogenase activity, we determined the subcellular localization of the enzyme and investigated its biochemical properties. Using density gradient centrifugation, we found that fatty aldehyde dehydrogenase activity was predominantly localized in the microsomal fraction in human liver. This fatty aldehyde dehydrogenase was solubilized from human liver microsomes and purified by chromatography on columns consisting of omega-aminohexyl-agarose and 5′-AMP-Sepharose 4B. The enzyme had an apparent subunit molecular weight of 54 000, required NAD + as cofactor, had optimal activity at pH 9.8, and was thermolabile at 47°C. Fatty aldehyde dehydrogenase had high activity towards saturated and unsaturated aliphatic aldehydes ranging from 6 to 24 carbons in length, as well as dihydrophytal, a 20-carbon branched chain aldehyde. In contrast, acetaldehyde, propionaldehyde, crotonaldehyde, glutaraldehyde, benzaldehyde, and retinaldehyde were poor substrates. The enzyme was inhibited by disulfiram, iodoacetamide, α, p-dibromoacetophenone, and p-chloromercuribenzoate. These results indicate that microsomal fatty aldehyde dehydrogenase is a distinct human aldehyde dehydrogenase isozyme that acts on a variety of medium- and long-chain aliphatic substrates.

Lactate dehydrogenase from the protozoan parasite, Trichomonas vaginalis

Lactate dehydrogenase (LDH) has been purified to near homogeneity from the parasitic protozoan, Trichomonas vaginalis, using ion exchange and affinity chromatographies. Two distinct peaks (P1 and P2) of LDH activity were eluted from DEAE-Sepharose, and LDH from each peak was purified separately. LDH from both peaks exhibited identical apparent subunit molecular weights of 39 kDa, as judged from SDS-PAGE and focused as single spots with identical pIs during 2D gel electrophoresis. In addition, activity staining for LDH activity on non-denaturing polyacrylamide gels of the crude homogenate yielded a single tight doublet, which was identical to the pattern seen when either P1 or P2 were analyzed under similar conditions. P1 and P2 exhibited dramatically different apparent K ms for pyruvate and lactate, but neither was affected by ATP, ADP, citrate, succinate, or fructose-1,6-diphosphate when assayed at near K m concentrations of pyruvate. In contrast to mammalian LDHs, the trichomonad enzyme could use 3-acetylpyridine NAD + as a substrate.

Purification and partial biochemical characterization of an S-type lectin from blastula embryos of Bufo arenarum

1. S-type lectin from Bufo arenarum embryos at blastula stage was purifed by affinity chromatography. The molecule is a dimer with equal-sized monomers and the apparent subunit molecular weight was found to be 14.5 kDa. 2. Analytical isoelectric focusing of the pure lectin showed an acidic pl of 4.7.3. Inhibition of the hemagglutination by mono- and oligosaccharides revealed a specificity for sugars bearing a β-galactoside configuration. 4. Crossreactivity studies between the blastula lectin and the one purified earlier from adult ovary performed by immunodotting, ELISA and immunoblotting showed that these lectins share many epitopes.

Purification and characterization of constituent androstenedione 15α-hydroxylase (Cytochrome P450 15αAD) from mouse liver : Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15α-hydroxylase (i.e. cytochrome P450 15αAD) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15α-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450 15αAD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7α- and 15α-hydroxylation of androstenedione, representing, respectively, >5% and >93% of the total metabolites. Polyclonal antibodies against cytochrome P450 15αAD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7α and 15α-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450 15αAD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450 15αAD was 53,000, and the protein appeared as a single band on sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450 15αAD is a member of the murine cytochrome P450 2c subfamily.

Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.

Purification and Characterization of Constituent Testosterone 2α-Hydroxylase (Cytochrome P4502α) from Mouse Liver

Hepatic microsomal testosterone/androstenedione 2α-hydroxylase (i.e., cytochrome P4502α was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2α-hydroxylase activity. The catalytic activity of the purified cytochrome P4502α, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2α-hydroxylation of testosterone and androstenedione; representing >96% and >92% of these respective metabolites. Polyclonal antibodies against cytochrome P4502α exhibited a dose-dependent and very selective inhibition of testosterone 2α-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P4502α fraction was 12.06 nmol/mg protein. The specific testosterone 2α-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P4502α was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P4502α is a member of the murine cytochrome P450 2d family.

Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions

The specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry were preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species.

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.

Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

A phosphatidylinositol (PI) 4-kinase was purified 25,000-fold from the cytosolic fraction of extracts from the yeast Saccharomyces cerevisiae. The purification consisted of an ammonium sulfate fractionation followed by chromatography on sulfonated-agarose (S-Sepharose), phosphocellulose, threonine-agarose, and quaternary amino (Mono Q), and sulfonated (Mono S) beads. Major contaminants in the purification, Hsc82 and Hsp82 (yeast homologs of the mammalian heat shock protein Hsp90), were eliminated by using a combination of molecular genetics (to construct a null mutation in HSC82), altered growth conditions (to minimize expression from the inducible HSP82 gene), and high ionic strength fractionation conditions (to remove the residual Hsp82). The purified enzyme had an apparent subunit molecular weight of 125,000, much larger than any other well characterized PI-4-kinase reported previously. Like mammalian PI-4-kinases, the yeast enzyme specifically phosphorylated PI on position 4 of the inositol ring and was stimulated by Triton X-100. However, activity was not inhibited by adenosine, a potent inhibitor of certain (type II) mammalian PI-4-kinases. The enzyme displayed typical Michaelis-Menten kinetics with apparent Km values of 100 microM for ATP and 50 microM for PI. To date, this yeast enzyme is the first soluble PI-4-kinase purified from any source.

Biochemical and antigenic properties of the Campylobacter flagellar hook protein.

The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.

Purification and properties of Plasmodium falciparum malate dehydrogenase

Asexual intraerythrocytic Plasmodium falciparum were shown to have a single isoenzyme of malate dehydrogenase. This malate dehydrogenase was purified to apparent homogeneity using a three-step purification protocol. The parasite malate dehydrogenase had an apparent subunit molecular weight of 32 kDa, a pH optimum of 7.0 for the reduction of oxaloacetate, and a sharp thermal transition between 40°C and 45°C. These characteristics distinguish P. falciparum malate dehydrogenase from both the cytoplasmic and mitochondrial malate dehydrogenase isoenzymes of humans. In addition, the resistance of the parasite malate dehydrogenase to substrate inhibition by oxaloacetate suggests that it is the cytoplasmic malate dehydrogenase isoenzyme. The apparent absence of mitochondrial malate dehydrogenase from asexual intraerythrocytic P. falciparum contributes to evidence indicating that the mitochondrion is undeveloped at this stage of the parasite's life cycle.

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED 50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 l crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31 32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.

Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.