Purification and characterization of alkaline ribonuclease inhibitor from normal and nephrotic rat kidney compared to inhibitor from rat liver

Kidney cortex, red medulla and white medulla were separated into nuclei, mitochondria, microsomal and 105000g supernatant fractions. Assay of RNAase (ribonuclease) activity at pH7.8 revealed that, for each subcellular fraction, activity was much greater in cortex than in red or white medulla; this was true for both free RNAase and total (free plus latent) RNAase. For example, the free RNAase activity in the 105000g supernatant of cortex was 5 and 8 times higher than in red and white medulla respectively. No latent RNAase activity was found in any particulate fraction. Latent supernatant RNAase activities (suggesting presence of bound RNAase inhibitor) were similar in cortex and medulla. The cortex supernatant contained minimal free RNAase inhibitor, whereas that of the red and white medulla showed about one-third and one-tenth respectively of the inhibitor activity measured in liver. Adrenalectomy did not change RNAase activity in any fraction nor the content of free RNAase inhibitor in the kidney supernatant, but did decrease the liver RNAase inhibitor content by 40%. In supernatants from mouse kidney, both free and total RNAase activities of both cortex and red medulla were similar to those of rat red medulla. Mouse cortex contained appreciably higher amounts of free RNAase inhibitor than rat cortex. The difference between the rat and mouse cortical RNAase activity and inhibitor content may help explain the relative ease with which satisfactory renal polyribosome profiles were obtained from mouse kidneys. Our results, as well as those of Kline & Liberti [(1973) Biochem. Biophys. Res. Commun.52, 1271-1277], showing that renal red and white medulla are more active than cortex in protein synthesis, are consistent with the hypothesis that the RNAase-RNAase-inhibitor system may participate in the regulation of protein synthesis.

Section: Discussioncontrasting

confidence: 95%

“…A possible explanation is that they purified the bound inhibitor. The difference between our results and those of Bishay & Nicholls (1973) is not attributable to the use of different strains of rats. In most of our studies we used Fischer rats, whereas they used Sprague-Dawley rats.…”

Section: Discussioncontrasting

confidence: 93%

Regional differences in ribonuclease content of rat and mouse kidney

Liu

Matrisian

1977

“…radioactivity 385Ci/mol, 0.56,Ci/mg of tRNA), containing 19 other unlabelled aminoacyl-tRNA species from Escherichia coli strain B, and other chemicals were obtained as described previously (Girgis & Nicholls, 1971Bishay & Nicholls, 1973;Innanen & Nicholls, 1973;Nicholls et al, 1974;Young & Nicholls, 1978). Myosin, actin and tropomyosin were gifts from Dr. A. Forer of York University.…”

mentioning

confidence: 99%

Studies of a factor from dystrophic mouse muscle inhibitory towards protein synthesis

Petryshyn¹,

Nicholls²

1978

A substance inhibitory to protein synthesis was purified from mouse skeletal muscle by gel filtration and ion-exchange chromatography, as well as by centrifugation on sucrose gradients. The molecular weight of the inhibitor, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 71000. The inhibitory activity was insensitive to ribonuclease A, deoxyribonuclease I and phospholipase C. It was sensitive to Pronase treatment but insensitive to heat-treatment and trypsin degradation. The present results, taken together with previous studies, indicate that the site of action of the inhibitor is not on the initiation phase of protein synthesis but rather at a step after the binding of aminoacyl-tRNA to ribosomes. The increased inhibitor activity found in dystrophic muscle is discussed.

“…Similar changes were found in kidney responding to growth-hormone administration (Girgis & Nicholls, 1973) or undergoing hypertrophy in response to folic acid (Nicholls et al, 1975). An increase in aminoacyl-tRNA binding also was observed during the increased synthesis of specific kidney proteins, such as the soluble ribonuclease inhibitor during aminonucleoside nephrosis and the microsomal proteins during the induction of kidney enzymes by DDT [1,1,1-trichloro-2,2-bis-(pchlorophenyl)ethane] and polycblorinated biphenyls (Girgis & Nicholls, 1971;Bishay & Nicholls, 1973;Nicholls & Markle, 1974;Cappon & Nicholls, 1974b;Cappon et al, 1976).…”

mentioning

confidence: 99%

Regeneration of renal proximal tubules after mercuric chloride injury is accompanied by increased binding of aminoacyl-transfer ribonucleic acid

McEwen

1976

Homogenates of rat kidney cortex obtained 1,3 or 14 days after a single injection of HgCl2 were used to prepare the post-microsomal pH5 supernatant fraction. The activity of this fraction for peptide synthesis from [14C]phenylalanyl-tRNA was significantly increased at 1 and 3 days, at which time the proximal tubules are regenerating [Cuppage & Tate (1967) Am. J. Pathol. 51, 405-429]. This increased activity could not be attributed to a decreased inhibitory activity, but was due to an increased aminoacyl-tRNA binding, i.e. elongation-factor-1 activity, in the supernatant fraction.