Subunit structure of thrombin-activated porcine factor VIII

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent denaturation of thrombin-activated porcine factor VIII

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.     

Coagulant properties of hybrid human/porcine factor VIII molecules.

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.     

Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation

Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance.     

A1 subunit-mediated regulation of thrombin-activated factor VIII A2 subunit dissociation

Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit

Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.     

Association of the factor VIII light chain with von Willebrand factor

Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.     

Enhanced biosynthesis of coagulation factor VIII through diminished engagement of the unfolded protein response

Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast, a significant proportion of porcine fVIII was localized to the Golgi indicating efficient transit through the secretory pathway. Overexpression of BiP, an integral UPR protein, reduced the secretion of human fVIII by 50%, but had no effect on porcine fVIII biosynthesis. In contrast, expression of BiP shRNA increased human fVIII expression levels. The current data support the model of differential engagement of UPR by human and porcine fVIII as a non-traditional mechanism for regulation of gene product biosynthesis.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.     

Molecular heterogeneity of the subclasses of islet-activating protein (pertussis toxin)-sensitive GTP-binding proteins in porcine thyroid tissue

From porcine thyroid cell membranes, we purified five GTP-binding proteins (G-proteins); Nos. 1 to 3 have 41-kDa alpha-subunits, and Nos. 4 and 5 have 40-kDa alpha-subunits. They were chromatographically (Mono Q) separable and served as specific substrates for islet-activating protein (pertussis toxin). G-proteins 1 and 2 were indistinguishable from porcine brain Gi1 with respect to three criteria, i.e., mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI of the ADP-ribosylated alpha-subunit, and immunoreactivity. G-protein 3 was identified as Gi3 by immunoreactivity. The SDS-PAGE and isoelectric focusing (IEF) analyses identified G-proteins 4 and 5 as being chromatographically heterogeneous subtypes of Gi2 in comparison with a pure porcine brain preparation. The IEF analysis also disclosed that each of the Gi1, Gi2, and Gi3 subspecies isolated in the present study has a minor component characterized by a slightly lower pI of its alpha-subunit. We conclude that porcine thyroid tissue contains at least Gi1, Gi2, and Gi3, and that each is made up of heterogeneous populations.     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Purification and some properties of Pseudomonas fluorescens lipase

Lipase (triacylglycerol lipase, EC 3.1.1.3) has been purified from Pseudomonas fluorescens wild strain by chromatography on DEAE-cellulose and octyl-Sepharose CL-4B. The yield was 21% and the specific activity of the purified enzyme 4780 U/mg protein. It showed a Mr of about 45 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is active over a wide pH range and at 50-55 degrees C.     

Potentiation of Thrombin Generation in Hemophilia A Plasma by Coagulation Factor VIII and Characterization of Antibody-Specific Inhibition

Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20–30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as “type I”) kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as “type II”) inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.     

Crystallization and preliminary X-ray study of horse pancreatic lipase

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.     

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.