Horacio A. Sancovich

Porphyrin biosynthesis: VI. Separation and purification of porphobilinogen deaminase and uroporphyrinogen isomerase from cow liver. Porphobilinogenase an allosteric enzyme

Abstract 1. 1.|Porphobilinogenase has been isolated and purified from cow liver and its components, porphobilinogen deaminase and uroporphyrinogen isomerase, have been separated from each other and purified. 2. 2.|The effect of NH 4 + was studied. The deaminase exhibited classical Michaelis-Menten kinetics in the absence or presence of NH 4 + , which at high concentrations behaved as a noncompetitive inhibitor of the deaminase. As expected from Hill plots, n = 1 both in the absence or presence of NH 4 + . Instead, when activity of porpho bilinogenase is plotted versus porphobilinogen concentration, sigmoid curves are obtained; but the presence of NH 4 + at different concentrations altered the kinetic parameters of this enzymic system, again showing normal kinetics. In addition, n values were found to be 2 for porphobilinogen per porphobilinogenase molecule and 1 in the presence of NH 4 + which behaves as a competitive inhibitor of the isomerase. Results are discussed in relation to the allosteric theories of monod et al. 1,2 and liver porphobilinogenase is proposed to be an allosteric protein. 3. 3.|The presence of an ultrafiltrable factor which stimulates uroporphyrinogen formation from porphobilinogen has been revealed.

Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats

The effect of hexachlorobenzene (HCB) (1 g/kg bw) administration for 4 weeks, on thyroxine (T4) and triiodothyronine (T3) metabolism was studied in Wistar rats. The effect on serum binding of T4 has also been studied. Animals were injected with a tracer dose of either labeled hormone and by examining serum L-125I-T4 and L-125-I-T3, kinetics of radiolabeled hormones metabolism were calculated. The T4 metabolic clearance (MCI) as well as the distribution space, were increased by 6 fold. Decreased serum T4 levels result from an increase both in deiodinative and fecal disposal in HCB-treated rats. 125I-T3 metabolism was slightly affected. The enhanced peripheral disposition of thyroxine appears to lead to increased thyroid function, as measured by augmented TSH serum levels and 125I-thyroidal uptake. Serum binding of T4 was not affected.

[19] Preparation of porphobilinogen

Publisher Summary This chapter illustrates the preparation of porphobilinogen (PBG). PBG is a normal intermediate in the biosynthesis of heme. An enzymatic method has been considered for the preparation of porphobilinogen with good yield using both δ-aminolevulinic acid and the enzyme aminolevulinic acid dehydratase. PBG can be isolated and purified by either of the two procedures of isolation and can be essentially identified by different methods. By paper chromatography, a biosynthetic PBG runs as a single spot having the same R f as that for the markers. A solution of biosynthetic PBG when mixed with an equal volume of Ehrlichs reagent gives maxima at 555 m μ and 525 m μ . PBG can be enzymatically converted into uroporphyrinogens by means of the deaminase–isomerase enzymatic complex.

The porphobilinogen deamise - uroporphyrinogen III cosynthetase system (porphobilinogenase) from bovine liver. Kinetic studies☆

The conversion of prophobilinogen (PBG) into uroporphyrinogens is enzymically catalysed by the porphobilinogenase enzyme system which is widely distributed [l-lo]. In plant tissue extracts [ 1 l] the formation of uroporphyrinogen I from PBG is brought about by PBG deaminase (Uroporphyrinogen I synthetase); in the presence of both PBG deaminase and uroporphyrinogen III cosynthetase (isomerase) uroporphyrinogen III, the physiological intermediate in haem biosynthesis, is formed. Here we report kinetic studies using purified PBG deaminase and porphobihnogenase. The effects of NH: ion concentrations on the type of uroporphyrinogen formed are also reported. The results indicate that bovine liver porphobilinogenase shows cooperative effects.

Hepatic indices of thyroid status in rats treated with hexachlorobenzene

The functional thyroid status of hexachlorobenzene (HCB)-treated rats was studied. HCB caused a depletion of serum thyroxine (T4 ), but did not change L-3,5,3’-triiodothyronine (T3) levels in serum of rats. The activities of the thyroid regulated mitochondrial enzyme L-glycerolphosphate dehydrogenase (LGPD) and cytosolic enzymes, malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and 6-phos-phogluconate dehydrogenase (6PGD) were assayed in livers of normal and HCB (100 mg/100 g bw) treated Wistar rats. Mitochondrial LGPD activity did not change significantly, however ME, 6GPD and G6PD were induced by HCB only in non-thyroidectomized animals. The absence of cytosolic enzymes induction in thyroidectomized rats treated with HCB indicates that HCB is not intrinsically thyromimetic. The induction of hepatic ME, G6PD and 6PGD activities in HCB thyroidectomized rats was dependent on the presence of thyroid hormone. The unchanged activity of mitochondrial LGPD in contrast to the increased activities of the cytosolic enzymes ME, G6PD and 6PGD is not consistent with a shift in functional thyroid status following HCB treatment.

Effect of in vivo administered hexachlorobenzene on epidermal growth factor receptor levels, protein tyrosine kinase activity, and phosphotyrosine content in rat liver.

In the present study, the effects of hexachlorobenzene (HCB) on epidermal growth factor receptor (EGFR) content of liver microsomes and plasma membrane, and on EGFR-tyrosine kinase activity in the microsomal fraction were investigated. In addition, we studied the parameters of the tyrosine kinase signalling pathway such as protein tyrosine kinase (PTK) activity and phosphotyrosine content in microsomal and cytosolic protein. To determine whether the observed alterations were correlated with a manifestation of overt toxicity, a single very low dose of HCB (1mg/kg body wt) and two much higher doses (100 and 1000 mg/kg body wt), the highest being toxicologically significant in that it reduced serum thyroxine (T(4)) and inhibited uroporphyrinogen decarboxylase (URO-D) (EC 4.1.1.37) activity, were tested. Our results demonstrated that liver microsomes of rats treated with HCB had higher levels of EGFR than untreated rats; treated rats also had less EGFR present in hepatocyte plasma membrane fractions than did untreated rats. HCB altered the phosphotyrosine content and protein phosphorylation of some microsomal and cytosolic proteins in a biphasic dose-response relationship. At the low dose, phosphorylation and phosphotyrosine content of several microsomal proteins were increased; however, these effects were diminished or reversed at the higher doses. Our results suggest that chronic HCB treatment produces a down-regulation of the EGFR and a dose-dependent increase in EGFR-tyrosine kinase activity in the microsomal fraction. This effect may contribute to the alteration of membrane and cytosolic protein tyrosine phosphorylation. The level of sensitivity encountered in our studies is extraordinary, occurring at 1/10 to 1/1000 the doses of HCB known to cause other toxicological lesions.

Porphyrin biosynthesis—immobilized enzymes and ligands—X. A novel approach to the study of the relationship between the quaternary structure of aminolaevulinate dehydratase and its activity

Abstract 1. 1. Evidence for dissociation, renaturation, re-association and re-hybridization of bovine liver aminolaevulinate dehydratase attached to Sepharose 4B is reported. 2. 2. When insolubilized enzyme was treated with 3 and 6 M urea, non covalently bound subunits were dissociated and detected in the eluate; these subunits can be re-associated into a soluble functioning enzyme with a specific activity close to that of the original pure soluble dehydratase preparation. 3. 3. After being washed with a renaturing buffer mixture, the matrix-bound subunits recovered a level of enzymatic activity equal to 50 and 20% of that of the immobilized native aminolaevulinate dehydratase. 4. 4. The reversibility of the dissociation process was investigated. Bound-subunits dehydratase can associate with nascent soluble bovine liver aminolaevulinate dehydratase subunits in situ. The product of such treatment, bound-re-associated enzyme, has the same activity as that of the original bound-dehydratase. The matrix-bound-dissociated bovine liver enzyme was also re-hybridized with soluble dehydratase subunits from E. gracilis . 5. 5. The apparent K m and optimum pH of the immobilized subunits were the same as those of the bound-octameric enzyme. 6. 6. A scheme is proposed, explaining the sequence of reactions leading from the bound-octameric dehydratase to the possible different derivatives, formed during the dissociation and re-association experiments.

Further Evidence for an Essential Histidyl Residue at the Active Site of Pig Liver 5-Aminolevulinic Acid Dehydratase

Photoxidation with methylene blue and rose bengal and chemical modification by diethylpryrocarbonate of pig liver 5-aminolevulinic acid dehydratase produced strong inactivation of the enzyme which was concentration dependent. Loss of enzyme activity by both photoxidation and ethoxyformylation was pH and time-dependent and protected by the presence of the substate and competitive inhibitors. The rate of inactivation was directly related to the state of protonation of histidyl groups, the unprotonated from being modified at a much faster rate than the protonated form. Plots of the pseudo-first order rate constants for 5-aminolevulinic acid dehydratase inactivation against pH resulted in typical titration curves showing inflection points at about pH 6.4 for methylene blue and rose bengal and 6.8 for diethylprocarbonate providing further and unequivocal evidence for the existence of critical histidyl groups at the active centre of the enzyme.

Uroporphyrin III: Its enzymic synthesis

Abstract An enzymic method has been developed for the preparation of uroporphyrin III using both porphobilinogen and porphobilinogenase from avian erythrocytes.

Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase.

5-Aminolevulinic acid dehydratase catalyses the self condensation between two molecules of 5-aminolevulinic acid, via a Schiff base, in which a lysine residue at its active site is proposed to be involved. The aim of the work was to further clarify the mechanism of this step in porphobilinogen biosynthesis. 5-Aminolevulinic acid dehydratase was purified 230-fold from pig liver, e -Aminolysil residues were identified by treating the enzyme with pyridoxal 5-phosphate. Schiff bases formed between either the substrate or pyridoxal 5-phosphate and the enzyme were stabilized by NaBH 4 reduction. Levulinate and pyruvate acted as competitive enzyme inhibitors. Pyridoxal 5-phosphate but not pyridoxamine 5-phosphate reversibly inhibited the enzyme activity, in a competitive fashion ( K i = 0.12 mM). After NaBH 4 treatment this inhibition became irreversible. The amount of labelled substrate bound to the enzyme after NaBH 4 reduction decreased in the presence of either pyridoxal 5-phosphate, levulinate or pyruvate. Enzyme elution profiles from Sephacryl S-300 showed that NaBH 4 treatment (1) in absence of substrate, did not induce any change on the enzyme, eluting as a typical 5-aminolevulinic acid dehydratase single peak (Mw 280,000), which overlapped with that of the enzyme activity; and (2) in the presence of labelled 5-aminolevulinate, had an additional peak eluting with a Mw of 140,000, without enzyme activity. This peak coincides in shape and elution volume with the radioactive one. These data suggest that pyruvate regulates pig liver 5-aminolevulinic acid dehydratase activity in vivo and that during catalysis, a tetrameric structure forms the enzyme-substrate complex. The results support the involvement of a critical e -aminolysil group at the active site of the enzyme.