Ernest Kun

[14] Stabilization of mitochondrial functions with digitonin

Publisher Summary The great diversity of intrinsic stabilities of various mitochondrial systems, especially those contributing to the intactness of the transfer capabilities of the inner mitochondrial membrane, poses special difficulties to experimentalists. The acceptor control index measured within short periods after isolation of mitochondria by conventional methods is a correct test of mitochondrial intactness, yet the specific activity of the adenosine triphosphate (ATP) synthetase system may vary greatly in various preparations exhibiting the same acceptor control index. Therefore, it is evident that remaining within the realm of oxidative phosphorylation, two diverse criteria of apparent functional intactness of the inner mitochondrial membrane can be obtained. This phenomenon is readily demonstrable if conventionally prepared mitochondria are incubated without substrates or cofactors at 30 ° C for 10–20 min and both acceptor control index and ATP synthetase are simultaneously monitored. The relatively slow mitochondrial processes play a significant role in experimental work when intracellular regulation of mitochondria is to be investigated with isolated mitochondria because functional intactness of these particles must be maintained for prolonged periods in vitro .

Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I).

The enzymatic transfer of ADP-ribose from NAD to histone H1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max. Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a = 1400versus 3–15, respectively). Mg2+ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg2+ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADP- ribosylation), thereby opening the possibility to study the physiologic function of PARP I.

Isolation and properties of a β-mercaptopyruvate-cleaving copper enzyme

1. 1. The distribution and isolation of β-mercaptopyruvate transsulfurase is described. The enzyme was isolated from rat liver by (NH4)2SO4 fractionation and repeated preparative electrophoresis. 2. 2. The enzyme was characterized by constant specific activity, copper content and its absorption spectrum. 3. 3. The mechanism of action of 2-mercaptoethanol and Na2SO3 on the enzymic formation of pyruvate from β-mercaptopyruvate was determined. While 2-mercaptoethanol acts as a reducing and stabilizing agent, SO3= reacts as a second substrate to accept S from β-mercaptopyruvate. 4. 4. The catalytically active group of the enzyme, which participates in the removal and transfer of S from the substrate, was identified as a thiol-disulfide-Cu system. A mechanism of transsulfuration based on analytical and kinetic measurements is described. This mechanism is restricted to the description of the reaction of the mercapto group of the substrate with the enzyme.

The reaction of β-mercaptop0ruvate with lactic dehydrogenase of heart muscle

Abstract A modified procedure for the preparation of pure ammonium β-mercaptopyruvate has been described. Evidence was obtained which indicates that the salt exists in solid state as the enol. LDH reversibly reduces β-mercaptopyruvate by DPNH. It is suggested that this reaction may be of physiological significance.

Fluorocitrate Inhibition of Aconitase: Relative Configuration of Inhibitory Isomer by X-ray Crystallography

The fluorocitrate isomer that is a strong inhibitor and inactivator of aconitase has been shown by x-ray crystallographic studies on the rubidium ammonium salt to have the configurations (1R : 2R) or (1S : 2S) 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylic acid. A possible mechanism for the action of fluorocitrate is proposed which involves the 1R : 2R isomer suggested from biochemical data.

Molecular interactions between poly(ADP‐ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding

The molecular interactions of poly(ADP‐ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the ‘global’ Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP‐ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the ‘global’ Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I–Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S‐transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the ‘C clamp’ model derived from X‐ray crystallography.

The interaction of Adenosine Diphosphoribosyl Transferase (ADPRT) with a cruciform DNA

Adenosine Diphosphoribosyl Transferase is a eucaryotic nuclear protein that catalyses the transfer of ADP-ribose moiety of NAD+ to itself and other cellular proteins. DNA is required for this post-translational modification process. We present novel evidence that Adenosine Diphosphoribosyl Transferase interacts with the base of plasmid pUC7 cruciform. We speculate that, unlike a sequence-specific DNA binding protein, the transferase may recognize an unusual DNA conformation.

The oxidation of l-lactate by liver mitochondria

Abstract A single NAD + -dependent LDH was found in liver mitochondria, localized in the intermembrane space. Maximal aerobic oxidation of l -lactate requires externally added NAD + . Apparent K m for NAD + is the same for the aerobic oxidation of l -lactate by mitochondria, as for the oxidation of l -lactate by NAD + in the presence of mitochondrial LDH. Mitochondrial LDH contains bound NAD + which remains associated with the enzyme protein during acrylamide gel electrophoresis. Dismutation between l -lactate and acetoacetate by mitochondria is greatly stimulated by NAD + and this reaction is inhibited by DNP, while dismutation between pyruvate and acetoacetate is not. Results are interpreted in terms of a possible mitochondrial path of l -lactate oxidation. This process may involve an energy-dependent transfer of reducing equivalents between l -lactate + LDH-bound NAD + (H) and enzyme systems of the inner membrane (and matrix).

Kinetics of ATP-dependent Mg2+flux in mitochondria

ATP-dependent Mg2+ accumulation in isolated mitochondria occurs predominantly in the matrix and inner membrane compartments. In mitochondria contaminated with lysosomes, the time course and magnitude of ATP-dependent Mg2+ accumulation are influenced by various cytoplasmic substances, besides substrates of the citric acid cycle. Removal of lysosomes by treatment of the mitochondrial preparation with low concentrations of digitonin, which does not damage the mitoplast, eliminates the modifying influence of cytoplasmic components on Mg2+ flux. In lysosome-free mitochondria, the kinetics of Mg2+ flux is dependent only on the concentration of ATP, of Mg2+, and on the availability of site specific reducing substrates of the electron transport system. Oligomycin at concentrations sufficient to inhibit phosphorylation coupled electron transport and ATP synthesis does not modify Mg2+ flux, which is dependent on added ATP. Site specific inhibitors of the electron transport system inhibit the augmenting effect of oxidizable substrates on Mg2+ uptake, even when electron transfer is inhibited by oligomycin. Atractyloside, by inhibiting the action of externally added ATP, diminishes Mg2+ flux. Ruthenium red is a powerful inhibitor of ATP dependent Mg2+ flux. Uncouplers not only inhibit Mg2+ uptake, but induce Mg2+ efflux. From the time course of Mg2+ flux, a first-order rate constant of egress of Mg2+ and other kinetic constants were calculated and a kinetic model was derived which describes the bi-directional movement of Mg 2+ in mitoplasts.

Inhibition of carcinogen-induced cellular transformation of human fibroblasts by drugs that interact with the poly(ADP-ribose) polymerase system: Initial evidence for the development of transformation resistance

Two types of interactions of 13 drugs with human fibroblasts were determined: (a) I 50 of nuclear poly(ADP‐ribose) polymerase, as assayed with isolated nuclei in vitro, and (b) the non‐toxic concentration of drugs that prevented carcinogen‐induced cell transformation of intact fibroblasts (RCF1). In general, RCF1 was much lower than I 50, and one antitransformer did not inhibit the enzyme in vitro, indicating that low‐affinity enzyme inhibitory sites appear to play no role in the mechanism of prevention of cell transformation. Two enzyme inhibitors, caffeine and 1‐methylnicotinamide, exhibited no antitransforming activity. Benzamide when applied in population doubling 1 induced resistance to cell transformation in population doubling 6 by carcinogens added at this stage.