Relationships between lead absorption and peripheral nerve conduction velocities in lead workers. (1/277)

The motor sensory, and mixed nerve conduction velocities of median and posterior tibial nerves were measured in 39 lead workers whose blood lead (PbB) concentrations ranged from 2 to 73 mug/100 g with anaverage of 29 mug/100 g. The PbB concentrations significantly correlated with the maximal motor nerve conduction velocities (MCV) and mixed nerve conduction velocities (MNCV) of the median nerve in the forearm and with the MCV of the posterior tibial nerve. Erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity correlated similarly with the MCV and MNCV of the median nerve in the forearm, and the 24-hour urinary lead excretion following the intravenous administration of CaEDTA (20 mg/kg) (lead mobilization test) correlated with the MNCV. But no parameter correlated with the sensory nerve conduction velocities. By multiple regression analysis, a combination of the three parameters of lead absorption was found to correlate significantly with the MCV and MNCV of the median nerve in the forearm. The MCVs of the median and posterior tibial nerves in lead workers were significantly delayed in the PbB range of 29-73 mug/100 g (mean 45), in the lead mobilization test range from 173 to 3,540 mug/day (mean 973), and the ALAD activity range from 4.4 to 19.4 u. (mean 14.0), respectively.  (+info)

The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid. (2/277)

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.  (+info)

Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex. (3/277)

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.  (+info)

Biosynthesis of delta-aminolevulinic acid from the intact carbon skeleton of glutamic acid in greening barley. (4/277)

The customary route in animals and bacteria for delta-aminolevulinic acid biosynthesis is from glycine and succinyl CoA, catalyzed by the enzyme delta-aminolevulinic acid synthetase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37]. Attempts to demonstrate this route in plants have been unsuccessful. Evidence is given for a new enzymic route of synthesis of delta-aminolevulinic acid in plants. This route involves the incorporation of the intact five-carbon skeleton of glutamic acid into delta-aminolevulinic acid. Demonstration of the new pathway in plants has been made by feeding specifically labeled [14C]glutamic acid to etiolated barley shoots greening in the light. In the presence of levulinate, a competitive inhibitor of delta-aminolevulinic acid dehydrastase [porphobilinogen synthase; delta-aminolevulinate hydro-lyase (adding delta-aminolevulinate and cyclizing); EC 4.2.1.24], delta-aminolevulinate accumulates. The delta-aminolevulinate formed was chemically degraded by periodate to formaldehyde and succinic acid. The C5 (formaldehyde) fragment was separated, as the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) derivative, from the C1-C4 (succinic acid) fragment. The C5 atom contained radioactivity predominantly derived from C1 of glutamic acid. Conversely, the labeled C3 and C4 atoms of glutamic acid were found primarily in the succinic acid (C1-C4) fragment of delta-aminolevulinate. This labeling pattern for delta-aminolevulinic acid is consistent with a biosynthetic route utilizing the intact five-carbon skeleton of alpha-ketoglutarate, glutamate, or glutamine, and is inconsistent with the delta-aminolevulinic acid synthetase pathway utilizing glycine and succinyl CoA as precursors.  (+info)

An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning. (5/277)

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.  (+info)

Genetic susceptibility to lead poisoning. (6/277)

Major strides have been taken in the regulation of lead intoxication in the general population, but studies using genetic markers of susceptibility to environmental toxicants raise the question of whether genes can make certain individuals more vulnerable to environmental toxins such as lead. At least three polymorphic genes have been identified that potentially can influence the bioaccumulation and toxicokinetics of lead in humans. The first gene to be discussed in this review is the gene coding for delta-aminolevulinic acid dehydratase (ALAD), an enzyme of heme biosynthesis, that exists in two polymorphic forms. The resulting isozymes have been shown to affect the blood and bone lead levels in human populations. The effects of ALAD in lead intoxication have also been studied in laboratory mice that differ in the genetic dose for this enzyme. The second gene reviewed here is the vitamin D receptor (VDR) gene. The VDR is involved in calcium absorption through the gut and into calcium-rich tissues such as bone. Recent findings suggest that VDR polymorphism may influence the accumulation of lead in bone. Finally, the third gene to be discussed here that may influence the absorption of lead is the hemochromatosis gene coding for the HFE protein. The presence of mutations in the HFE gene leads to hemochromatosis in homozygotic individuals. Because of the associations between iron and lead transport, it is possible that polymorphisms in the HFE gene may also influence the absorption of lead, but this has not yet been studied. More studies will be needed to define the role of these genes in lead intoxication.  (+info)

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates. (7/277)

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.  (+info)

Alcohol and porphyrin metabolism. (8/277)

Alcohol is a porphyrinogenic agent which may cause disturbances in porphyrin metabolism in healthy persons as well as biochemical and clinical manifestations of acute and chronic hepatic porphyrias. After excessive consumption of alcohol, a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria is observable, which can become persistent in cases of alcohol-induced liver damage. Nowadays, the alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria) are considered to be molecular regulatory diseases, in contrast to non-acute, chronic hepatic porphyria, clinically appearing as porphyria cutanea tarda (PCT). Porphyrins do not accumulate in the liver in acute porphyrias, whereas in chronic hepatic porphyrias they do. Thus, chronic hepatic porphyria is a porphyrin-accumulation disease, whereas acute hepatic porphyrias are haem-pathway-dysregulation diseases, characterized in general by induction of delta-aminolevulinic acid synthase in the liver and excessive stimulation of the pathway without storage of porphyrins in the liver. The clinical expression of acute hepatic porphyrias can be triggered by alcohol, because alcohol augments the inducibility of delta-aminolevulinic acid synthase. In chronic hepatic porphyrias, however, which are already associated with liver damage, alcohol potentiates the disturbance of the decarboxylation of uro- and heptacarboxyporphyrinogen, which is followed by a hepatic accumulation of uro- and heptacarboxyporphyrin and their sometimes extreme urinary excretion. Especially in persons with a genetic deficiency of uroporphyrinogen decarboxylase, but also in patients with the so-called sporadic variety of PCT, alcohol is able to transform an asymptomatic coproporphyrinuria into PCT. Alcohol has many biochemical and clinical effects on porphyrin and haem synthesis both in humans and laboratory animals. Ethanol suppresses the activity of porphobilinogen synthase (synonym: delta-aminolevulinic acid dehydratase), uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase, whereas it induces the first and rate-limiting enzyme in the pathway, delta-aminolevulinic acid synthase and also porphobilinogen deaminase. Therefore, teetotalism is a therapeutically and prophylactically important measure in all types of hepatic porphyrias.  (+info)