Biomedical subjects

H Wombacher

Publications and source records attributed to H Wombacher.

At least 19 recordsLinked to original sources

PubMed1986

Purification of an enzyme aggregate containing 3',5'-cyclic-nucleotide phosphodiesterase and nucleotidase.

Several steps of purification (octyl-Sepharose chromatography, Blue Sepharose 6B chromatography and sucrose density gradient centrifugation) led to a highly purified aggregate of the enzymes, 3',5'-cyclic-nucleotide phosphodiesterase (PDE) and nucleotidase. The purified enzyme aggregate showed an S value of 7.3 (SE +/- 0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) revealed two proteins near 67 and 60 kDa. Dissociation of the 7.3 S enzyme aggregate showed a 3.6 S PDE form and a nucleotidase form at 4.2 S. Additionally, higher S value forms of the nucleotidase up to 17 S have been observed. Apparently, they had formed by self-association. SDS-PAGE of the 17 S nucleotidase form showed only one band at 67 kDa. This was taken as evidence for the homogeneity of the 17 S nucleotidase form and the self-association of the nucleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protein of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1983

Molecular compartmentation by enzyme cluster formation. A view over current investigations.

Current investigations in different fields of cellular metabolism focus on the phenomenon of molecular compartmentation as an essential part of metabolic control. This type of compartment without surrounding membranes arises from enzyme cluster formation in the cell. The organization of the enzymes ranges from very loose, non-covalent aggregations, sometimes only transiently associated - dependent on metabolic or developmental state of the cell - to the very fixed, even covalently linked structures. These organized multienzyme systems produce a chemical microheterogeneity concerning the metabolite concentrations in the cell. Molecular compartmentation is the description of this chemical microheterogeneity in a biological term.

Animals↗

PubMed1982

Theophylline effect on the cyclic AMP degrading multienzyme sequence.

Membrane-bound 3'.5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) is closely associated physically with nucleotidase and deaminase, thus forming an enzyme cluster of unique catalytic behaviour [H. Wombacher, Archs. Biochem. Biophys. 201, 8 (1980)]. This multienzyme cluster, which was found in the microsomal fraction of beef adrenal cortex, catalyses the degradation of cyclic AMP, via AMP and adenosine, to inosine. The present study shows how theophylline, a well-known inhibitor of the phosphodiesterase, acts on the membrane-bound multienzyme sequence. The findings were as follows. Firstly, as expected, theophylline inhibited the phosphodiesterase competitively. In particular, the high-affinity enzyme was inhibited by mM concentrations of theophylline. Phosphodiesterase activity was tentatively ascribed to two enzymes, one with a low Km [0.3 microM], one with a high Km [60 microM]. Secondly, theophylline inhibited the nucleotidase activity to a great extent. A detailed kinetic analysis showed the inhibition to be hyperbolic noncompetitive (alpha = 1, beta = 0.35 and Ki = 0.25 mM). Thirdly, theophylline did not inhibit the deaminase activity of the multienzyme sequence. A model of theophylline inhibition is suggested explaining how an effector could modulate the kinetic behaviour of an enzyme cluster by acting at a single allosteric site. Finally, in view of the existence of the cyclic AMP degrading multienzyme sequence and the effect of theophylline on it, the possibility is discussed that physiologically active adenosine is derived from cyclic AMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1981

Kinetic and inhibition studies of Bacillus cereus beta-lactamase using a spectrophotometric method.

The use of a spectrophotometric method is reported for the characterization of a beta-lactamase (EC 3.5.2.6) from Bacillus cereus. Absorption coefficients of the mercaptides of various penicillins were determined with this method. The enzyme was kinetically characterized using penicillins. Inhibition studies with Bacillus cereus beta-lactamase and various penicillins showed a substrate type of inhibition, indicating an additional binding site for substrates without catalytic activity. The dissociation constant of this binding site was determined and the influence of this binding site upon the catalytic activity is discussed. Studies with beta-lactamase-stable penicillins as inhibitors and various penicillins showed different types of inhibition, which indicated the presence of an additional catalytically inactive binding site. Experiments with clavulanic acid, a beta-lactamase inhibitor without remarkable intrinsic antibacterial activity, showed a mixed type of inhibition. Based on the hypothesis for the existence of more than one substrate binding site on the enzyme, clavulanic acid was found to be bonded to both the catalytic active and the catalytic inactive binding site.

Bacillus cereus↗

PubMed1981

Membrane milieu is regarded as the native environment of the cyclic AMP degrading enzyme cluster.

The phenomenon of kinetic advantage of nucleoside formation from cyclic AMP, via the intermediate 5'AMP has been observed in the microsomal fraction after subcellular fractionation of beef adrenal cortex tissue. It was explained by the existence of a multienzyme sequence previously evidenced [H. Wombacher, 1982, Arch. Biochem. Biophys. 201, 8-19]. In the present study a similar enzyme cluster was prepared from the soluble fraction of the cell homogenate after two steps of gel-chromatography. An elusive channeling of cyclic AMP degradation could be disclosed. The time course reaction of cyclic AMP degradation to the nucleosides, adenosine and inosine, via 5'AMP as an intermediate compared with the time course reaction of 5'AMP hydrolysis to the nucleosides, adenosine and inosine, under otherwise identical conditions showed that the nucleoside formation from cyclic AMP was faster after the lag phase of the reaction sequence. This kinetic advantage effect, however, was much less pronounced than to be seen in the membrane-bound multienzyme sequence. For an analysis of the influence of the environmental conditions on the activity of both enzyme cluster forms they were treated by chaotropic agents, detergents and ultrasonic power. Common to all results was: the activity of the membrane-bound enzyme cluster is highly stable in comparison with the soluble form. On basis of these and previous findings a hypothesis is suggested explaining the similarities between the membrane-bound enzyme cluster and the soluble form. Thus, the soluble enzyme cluster form is considered a partially preserved form of the membrane-bound form arisen from the cell homogenization process and/or vice versa the soluble form might present a pro-form of the membrane-bound enzyme cluster, and the most stable and active assembly has to be yet first membrane-triggered.

Adenosine Monophosphate↗

PubMed1980