Liver ferrochelatase from normal and hexachlorobenzene porphyric rats. Studies on their properties

• 1.1. The aim of the present work is to shed light on the way of action of hexachlorobenzene (HCB) on hepatic ferrochelatase the mitochondrial enzyme which catalyzes the last step of haem biosynthetic pathway.
• 2.2. Some properties of this enzyme from normal and HCB porphyric rat liver were studied.
• 3.3. The present findings indicate that HCB treatment would modify the configuration of the enzyme perhaps allowing the active center of the porphyric ferrochelatase to be more exposed.
• 4.4. As a consequence it would show: (a) its higher affinity for the iron; (b) the shorter time necessary to form the intermediate enzyme-substrate, reflected both by the existence of a shorter lag and consequently a shorter pre incubation time.
• 5.5. However this modification elicited by the fungicide does not alter the submitochondrial distribution of the enzyme nor the optimal conditions for its measurement.

     

Liver porphyrinogen carboxylase in hexachlorobenzene porphyric rats. Studies with intermediate porphyrinogens of series III and with uroporphyrinogen I

The present work studies the action of hexachlorobenzene (HCB) on the decarboxylation of uroporphyrinogen (Urogen) I and III and also on the decarboxylation of intermediate porphyrinogens of series III under different conditions using liver of normal and porphyric rats as enzyme source. The same enzyme is involved in the Urogen decarboxylation of both isomeric series I and III and catalyses the four steps in both cases. HCB affects all of them. HCB blocks the four steps of Urogen III decarboxylation to the same degree, as a function of intoxication time. HCB leads, in general, to an increase in the efficiency (Km/Vmax) of the porphyric system. These data can be interpreted as a reaction of the organism to overcome the enzymatic blockade.     

Heme content of normal and porphyric cultured skin fibroblasts

Partial deficiencies in enzyme activities of the heme biosynthetic pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients with protoporphyria (PP) and acute intermittent porphyria (AIP). We have quantitatively and qualitatively assessed the heme and free porphyrin content in cultured PP, AIP, VP (variegate porphyria, in which an enzymatic deficiency has not been identified), and normal skin fibroblasts during routine culture conditions in order to assess the overall metabolism of heme in these cells. The total heme concentration was not significantly different between control and porphyric lines; 189 +/- 15 pmoles/mg protein (mean +/- SEM) in controls, 154 +/- 17 in PP, 175 +/- 20 in AIP, and 181 +/- 81 in VP. The hemoprotein difference spectra were similar in all lines. Free porphyrins were not detected in any of the disorders. Despite partial deficiencies in enzyme activities of the heme pathway, porphyric fibroblasts thus maintain normal heme content during routine culture conditions without detectable porphyrin accumulation.     

Hexachlorobenzene porphyria and hexachlorobenzene catabolism in rats

Gas-chromatographic examinations were made on the amounts of hexachlorobenzene accumulating in the liver and fatty tissue of rats chronically poisoned with a diet containing 0.2 % hexachlorobenzene, and on the amounts of hexachlorobenzene and pentachlorophenol excreted with the urine and the faeces in the course of the poisoning. The results indicated a constant rise in the hexachlorobenzene levels in these tissues. Pentachlorophenol formed in the catabolism of hexachlorobenzene appeared in increasing concentration in both the urine and the faeces from the commencement of the poisoning. After the 5th–6th week of poisoning, the presence of other apolar and polar products in the excretions was also markedly enhanced. After a single dose of hexachlorobenzene /0.2 g/animal/, of all the decomposition products only pentachlorophenol was produced in high concentration, showing that this is a primary catabolite. A hypothesis is put forward as to the possibility of a role being played in the mechanism of action of hexachlorobenzene by a membrane permeability change.     

Mechanism of tissue-selective drug action in the cardiovascular system

Analysis of the human genome project tells us that there may be as few as 3000 genes that are likely to be good drug targets. Although the number of targets is still very large, these data have been interpreted by some to mean that the pharmaceutical industry may someday run out of novel drug targets. Despite the doom and gloom of such analysis, there is considerable reason for optimism. Drugs may exhibit selectivity of action beyond that predicted by target expression alone. Drugs that act at a single molecular target may have very different pharmacology and, as a result, different therapeutic uses. Three well-characterized model systems are highlighted to illustrate this point. The first model system is exemplified by nifedipine and verapamil, both of which act on L-type calcium channels. Both drugs are used to treat hypertension, but only verapamil can be used to produce atrioventricular block in patients with atrial fibrillation. The second model system describes the therapeutic exploitation of unusual conditions that occur in the ischemic myocardium to produce drugs that are more effective for suppressing ischemia-induced arrhythmias. The third model system discusses the mechanisms through which phosphodiesterase-5 (PDE5) inhibitors act selectively to facilitate penile erection while having little effect in the non-penile vasculature that also expresses PDE5.     

Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.

The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.     

Liver protein synthesis. Molecular weight distribution of pulse-labeled polypeptide chains in normal and thyroidectomized rats.

Equations are presented for determination of elongation rate in vivo for a heterogenous population of polypeptide chain molecular weights. The distribution of pulse-labeled polypeptide chains in rat liver deoxycholate-soluble protein has been obtained by sodium dodecyl sulfate-gel electrophoresis and used to compute a theoretical curve for determination of synthesis time of a 50000 mol. wt. polypeptide chain (tc50). Values of tc50 for normal and thyro-parathyroidectomized Long-Evans male rats were 1.2 and 1.75 min, respectively, representing protein synthetic rates of about 7.5 and 5.1 mg protein/g liver/h. No difference in the molecular weight profile of liver polypeptide chains on the basis of labeling or Amido-black staining was observed between the two groups. The distributions of radioactivity before and after secretion of labeled plasma protein are compared. The role of protein-synthetic rate in the changing enzyme levels associated with thyroid hormone is discussed.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action.

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.     

Mechanism of action of deoxyribonuclease II from human lymphoblasts.

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme. Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I.     

Biological action and binding sites for vasopressin on the mesenteric artery from normal and sodium-depleted rats

We have recently demonstrated specific binding for 3H-arginine8-vasopressin (3H-AVP) to high affinity sites on membranes of rat mesenteric arteries. We have now measured the biological activity of this peptide (AVP) and analogues on the perfused rat mesenteric artery. There was a close relationship between the ED50 of agonists or the pA2 of antagonists on the perfused tissue and the relative potency (IC50) of analogues for displacing 3H-AVP from the membrane preparation. The ED50 measured was 67 +/- 7 ng for AVP and 7.2 +/- 1.1 microgram for oxytocin. In sodium-depleted rats we have observed an increase (27%) of the maximal response to AVP with no significant change in ED50 (from 2.8 +/- 1.0 X 10(-8) M to 1.3 +/- 0.2 X 10(-8) M). On the membrane preparation, the number of binding sites for 3H-AVP was increased from 71 +/- 17 fmole/mg protein (Kd 3.5 +/- 0.5 nM) to 115 +/- 10 fmole/mg protein (Kd 4.8 +/- 0.3 nM) in the sodium-depleted rat by comparison to control animals. These results suggest that AVP and its analogues interact in a similar manner in the in vitro perfused rat mesenteric artery and with the membrane receptors isolated from the same tissue. Receptors for AVP are increased in the mesenteric vascular bed by sodium depletion.     

Mechanism of fluoroquinolone action.

When fluoroquinolones bind to gyrase or topoisomerase IV in the presence of DNA, they alter protein conformation. DNA cleavage results with diminished religation, so the enzymes are trapped in ternary complexes with drug and cleaved DNA. Preferential localization of gyrase ahead of replication forks and topoisomerase IV behind them causes fluoroquinolone-mediated complexes with the two enzymes to have different physiological consequences.