Distamycin inhibition of topoisomerase I-DNA interaction: a mechanistic analysis.

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA).(dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was unessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable band. Alternative modes of inhibition are discussed.     

Zinc-dependent angiotensin-converting enzyme in reactions with various enzymes

The effects of Zn2+ on the activity of the angiotensin-converting enzyme from bovine lung towards the substrates, FA-Phe-Gly-Gly and Cbz-Phe-His-Leu, have been studied. At pH below 7.0 zinc ions added to the reaction mixture increase the enzyme activity; this stimulating effect is changed to inhibition with a further rise in Zn2+ concentration. It was shown that the dissociation constant for the enzyme--Zn2+ complex and the "optimal" concentrations of Zn2+ needed for the manifestation of the maximal enzymatic activity depend on the nature of the substrate at all pH values studied.     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.     

Kinetic characterization of GABA-transaminase from cultured neurons and astrocytes

The enzymatic mechanism and the kinetic parameters of GABA-transaminase extracted from cultured mouse cerebral cortex neurons and astrocytes were studied. Neuronal as well as astrocytic GABA-transaminase obeyed a bi bi ping-pong reaction mechanism. The estimated K_m-values for -ketoglutarate and GABA were significantly lower for astroglial GABA-transaminase compared to the neuronal enzyme suggesting a possible existence of cell specific isozymes of GABA-transaminase. The observed enzymatic mechanism and the magnitude of the estimated kinetic parameters imply that GABA-transaminase synthesized in the two types of cultured neural cells is mechanistically and kinetically equivalent to the enzyme synthesized in the brainin vivo.