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Biomedical subjects

B E Fischer

Publications and source records attributed to B E Fischer.

26 records · Page 2Linked to original sources
PubMed1990

Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator.

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of the extracellular lipase of Acinetobacter calcoaceticus 69 V.

The extracellular lipase of Acinetobacter calcoaceticus 69 V was purified by hydrophobic interaction chromatography to homogeneity as suggested by gel electrophoretic analysis. The lipase existed as a high molecular complex of about 300 kDa, with a subunit molecular weight of 30.5 kDa being obtained by SDS-PAGE. The hydrodynamic molecular radius obtained by gel electrophoresis was 3.27 nm. The lipase had an isoelectric point of 5.5 and was stimulated by additions of deoxycholate. The activation energy for the hydrolysis of p-nitrophenyl palmitate was 39.9 kJ mol-1. Tri-, di- and monoacylglycerols were hydrolyzed. Hg2+ and p-hydroxymercuribenzoate inhibited the enzyme activity at very low concentrations. One sulfhydryl group was found per molecule of lipase.

Acinetobacter↗
PubMed1986

Localization of hydrolytic enzymes in Acinetobacter calcoaceticus.

The localization of hydrolytic enzymes, phosphatase, esterase, lipase and palmitoyl-CoA hydrolase was analysed in the cytosol, cytoplasmic membrane, periplasmic fraction, outer membrane and culture supernatant in dependence on the growth rate of the bacteria. The unspecific phosphatase was found to be a cytosolic enzyme. A lipase was the only extracellular enzyme detected. The results pointed to a secretion of the lipase into the culture medium via cytoplasmic and outer membrane. The palmitoyl-CoA hydrolase was found to be attached to the outer membrane, but activities were also detected in the periplasmic fraction. Unspecific esterolytic activities were mainly measured in the cytosol and in the cytoplasmic membrane.

Acinetobacter↗
PubMed1980

Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) and adenosine 5'-triphosphate (ATP).

The acidity constant of protonated 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) has been measured. The influence of hydroxo groups on the basicity of Bistris and related bases is discussed. The interaction of Bistris with the metal ions (M2+) Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 1.0 M, KNO3; 25 degrees C) and the stability constants of the M(Bistris)2+ complexes were determined. Unexpectedly Ca(Bistris)2+ is the most stable among the alkaline earth ion complexes (log KCaCa(Bistris) = 2.25; the corresponding values for the Mg2+, Sr2+ and Ba2+ complexes are 0.34, 1.44 and 0.85, respectively). The ions of the 3d series follow the Irving-Williams sequence: log KMnMn(Bistris) = 0.70, for Cu2+, 5.27 and Zn2+ 2.38. Ternary complexes containing ATP4- as a second ligand were also investigated: the values for delta log KM (= log KM(ATP)M(ATP)(Bistris) -log KMM(Bistris) are in general negative (e.g. delta log KCa = -0.40 or delta log KCu = -1.65), thus indicating that the interaction of Bistris with M(ATP)2- is somewhat less pronounced tan with M2+. However, even in mixed-ligand systems, complex formation may still be considerable, hence great reservations should be exercised in employing Bistris as a buffer in systems containing metal ions. Moreover, in several cases delta log KM is relatively high [for Mg2+-ATP4- -Bistris even positive], indicating some cooperativity between the coordinated ligands, possibly hydrogen-bond formation. Distributions of the complexes in dependence on pH are given, and the structures of the binary M(Bistris)2+ and the ternary M(ATP) (Bistris)2- complexes are discussed. The participation of Bistris hydroxo groups in complex formation is evident.

Adenosine Triphosphate↗
PubMed1979

Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5'-triphosphate (ATP).

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.

Adenosine Triphosphate↗
PubMedDate not supplied

Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity.

Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.

Biological Assay↗