Glyceraldehyde-3-phosphate dehydrogenase mRNA. Activity and amount in dystrophic hamster muscle.

The activity and amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in muscle of young dystrophic hamsters was reduced to approximately half the level found in control animals. No changes in brain or liver enzyme activity were found. Several other glycolytic enzyme activities and creatine kinase activity in muscle were unchanged, except for modest decreases in aldolase and pyruvate kinase. To assess the synthesis of glyceraldehyde-3-phosphate dehydrogenase, the poly(A)+ RNA was isolated from muscle polysomes of dystrophic and control animals and its activity was assessed in an mRNA-dependent translation system. The translatability of the mRNA for GAPDH found in the dystrophic muscle preparations also was half of that found in the control muscle preparations. Decreases were also found in the translatability of mRNA for tropomyosin.

Translation of muscle mRNA in rats following acute exposure to Pb2+ or Cd2+.

The hindleg muscle of rats was studied 2 days following the i.p. administration of 0.5 mg Pb2+ /100 g body wt or 0.12 mg Cd2+ /100 g body wt or both Pb2+ and Cd2+. The incorporation of [14C]leucine into proteins was measured using mRNA obtained from the muscle polysomes. The translatability of this poly(A)+ RNA in a mRNA-dependent reticulocyte lysate was elevated similarly in each of the preparations from heavy metal treated rats compared to control rats. Evidence for increased mRNA activity for glyceraldehyde-3-phosphate dehydrogenase and actin was obtained.

Translatability of rat kidney mRNA after mercury administration.

Young male rats received an intraperitoneal injection of 0.5 mg HgCl2/kg body weight and 16 h later the kidneys were removed and homogenized to prepare the polysomal fraction from which the poly(A)+ RNA was obtained. The activity of this fraction was assessed by translating the poly(A)+ RNA in a mRNA-dependent rabbit reticulocyte lysate and the activity was markedly elevated relative to preparations from control rat kidneys. The incorporation of labelled leucine and cysteine, but not phenylalanine, into a low molecular weight protein (approximately 10 000 as judged by denaturing polyacrylamide gel electrophoresis) accounted for the increased mRNA activity. The mobility during electrophoresis of the denatured labelled product and carboxymethylated product, as well as their acidic isoelectric points, provided evidence that it is metallothionein mRNA which exhibits increased translatability in preparations derived from mercury-treated rats.

Does lead exposure influence liver protein synthesis in rats?

The liver and serum of rats was studied 18 hr following the i.p. administration of 0.5 mg Pb2+/100 g body wt. The incorporation of [14C]leucine into proteins was measured in ribosomal and mRNA fractions incubated in vitro and was markedly stimulated in the Pb2+ treated rats. Similar results were found when the proteins of liver and serum were labelled by the injection in vivo of [14C]leucine. The increased mRNA activity appears to be chiefly for acid serum proteins of approx. Mr 44,000, 41,000, 33,000 and 18,000.

Kidney urokinase activity following acute exposure to lead.

Urokinase activity was measured in kidney homogenate fractions obtained 2 days after injecting rats with 0.5 mg Pb2+/100 g body weight. The activity was higher in the membrane-containing fractions than in the soluble supernatant fractions and was markedly higher in the preparations derived from the lead-treated rats. The kidney poly(A)+ RNA was obtained from these animals and translated in a rabbit reticulocyte lysate system. In the preparations obtained from the lead-treated rats, there was an increased synthesis of a protein, believed to be urokinase, of pl 8.6 and molecular weight (Mr) 45,000.

The activity of membrane enzymes in homogenate fractions of rat kidney after administration of lead.

Rats received one or two consecutive daily ip injections, each 0.5 mg Pb2+/100 g body weight, and the kidneys were studied 48 or 24 hr, respectively, after the injection. Renal brush border preparations from Pb2+-treated rats exhibited significant decreases in the activity of alanine aminopeptidase and gamma-glutamyl transpeptidase, greater after two injections, yet the amount of brush border protein remained unchanged. Moreover, the activity of alkaline phosphatase in the brush border was significantly increased after Pb2+. No significant changes in urine volume, urinary protein, or enzymes could be detected in these experiments. The enzymatic changes observed in the brush border after acute exposure to Pb2+ contrasted with those after exposure to Hg2+ where both the structure and enzymatic functions were severely damaged and after exposure to Cd2+ where enzymatic alterations were not accompanied by cytological changes.

Translation of mRNA from rat kidney following acute exposure to lead.

1. Two days after the administration of Pb2+ (0.54 mg/100 body wt) to rats, the kidneys were removed and homogenate fractions obtained. 2. The machinery for peptide synthesis exhibited increased activity in the kidney preparations from lead-treated rats. 3. The poly(A)+ RNA was obtained from the polysome fraction and translated in a mRNA-dependent reticulocyte lysate system. 4. The translatability of the poly(A)+ RNA was similar using the preparations obtained from control and lead-treated rats except for proteins of approx molecular weight of 50,000 which contained increased amounts [14C]leucine in the fractions derived from lead-treated rats. 5. The results are compared with those after acute Cd2+ toxicity where poly(A)+ RNA exhibited increased translatability for low molecular weight metallothioneins.

Translation of rat kidney mRNA after cadmium administration.

1. Twenty-four hours after the administration of Cd2+ (11 mumol/kg body weight) to rats, the kidneys were removed and the RNA was extracted from the polysomes and used to prepare poly(A) RNA. 2. The poly(A)+ RNA was translated in rabbit reticulocyte lysates containing different labelled amino acids as precursors and the resultant proteins were separated by polyacrylamide gel electrophoresis. 3. The labelling of the proteins was similar using poly(A)+ RNA obtained from control and Cd2+ treated rats except for two proteins. 4. Regardless of labelled precursor used, proteins of mobility in sodium dodecylsulphate electrophoresis of mol. wt 50,000 contained approx twice as much radioactivity using the RNA from the kidney of treated rats. 5. Using labelled leucine, lysine, and cysteine, but not labelled phenylalanine or histidine, proteins of mobility in sodium dodecylsulphate electrophoresis of mol. wt 10,000 contained approx twice as much radioactivity using the RNA from the kidney of the Cd2+ treated rats. These results and the results following carboxymethylation of the proteins prior to electrophoresis, together with the results from co-electrophoresis of the products [125-I]-labelled liver metallothionein support the view that the poly(A)+ RNA contains kidney mRNA for metallothionein.

The effect of cadmium on kidney ribosomes.

Rats received 2.6 mg/kg CdCl2 24 h prior to removing the kidneys and preparing the ribosomal fraction. The amount of ribosomal RNA was decreased 28% and there was a significant increase in bound ribosomes relative to free ribosomes following cadmium. The activity of these ribosomes from the kidney of cadmium-treated animals was nearly doubled either using endogenous mRNA or poly(U), together with elongation factors prepared from control rat liver. When ribosomal subunits were prepared and recombined in a cell-free assay system, the increased ribosomal activity was located in the 60S subunit. In chronic experiments for 2.5 or 11 weeks, cadmium chloride was administered in the drinking water at 100 or 250 ppm. The ribosomal RNA concentration was not decreased at 100 ppm for 2.5 weeks, yet the activity of the ribosomes was increased about 50%. AT 250 ppm for 2.5 weeks or 100 ppm for 11 weeks the ribosomal RNA concentration was decreased 15% and 7%, respectively, while the activity of the ribosomes was once more increased by about 50%. The increase in ribosomal activity after cadmium chloride are discussed in relation to the reported effects of cadmium chloride and of mercuric chloride on renal metallothionein and on renal tubules.

Peptide elongation in rat kidney after cadmium administration.

Rats received two injections (each 2.6 mg/kg body wt.) of CdCl2, and the kidneys were removed 24 h later. Postmicrosomal supernatant fractions of the homogenized kidneys were used as a source of elongation factors 1 and 2 in assays for [14C]phenylalanyl-tRNA binding to ribosomes and for peptide-bond synthesis. After purification of these preparations by precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200 and G-100, elongation factor 1 activity was significantly increased. A significant increase in the activity of purified elongation factor 2 was also found. The results are discussed in relation to the reported effects of CdCl2 and of HgCl2 on renal tissue.