Paul Bartlett

Separation of glomerular basement membrane substances by sodium dodecylsulfate disc gel electrophoresis and gel filtration.

Abstract Beef glomerular basement membranes, solubilized by sodium dodecylsulfate and β- mercaptoethanol , were examined by disc gel electrophoresis and gel filtration. The beef glomeruli were sonicated in either 0.15 m NaCl or 1.0 M NaCl, and the basement membranes sedimented at 1300 × g . Disc gel electrophoresis in sodium dodecylsulfate showed that the 1.0 M preparation contaiend predominantly high molecular weight components; the 0.15 M preparation contained moer low molecular weight components. Different amounts of hydroxyproline and DNA were found in the two membrane preparations. Other subunits were obtained when basement membrane was solubilized in 8 M urea or 7 M guanidine thiocyanate. Gel filtration of solubilized glomerular basement membrane was performed on 6% Agarose in sodium dodecylsulfate-phosphate. Partial separation of teh components observed in the disc gel electrophoresis was achieved. Protein and carbohydrate were present in all fractions.

Mechanism of aminonucleoside-induced nephrosis in the rat. III. Kidney mitochondrial phosphorylation and dephosphorylation activity.

Summary Results of time-course studies of the development of a significant reduction in disappearance of inorganic phosphate, associated with oxidation of succinate in kidney mitochondria of aminonucleoside-nephrotic rats, clearly indicate the establishment of this effect as early as the 4th day during the induction of the nephrotic syndrome, and prior to onset and development of massive proteinuria. Oxygen uptake was not similarly altered until the 6th day of aminonucleoside administration and occurred in parallel with onset of proteinuria. Under experimental conditions closely resembling those employed in the oxidative phosphorylation studies, no significant differences were noted in dephosphorylation of ADP, ATP, and G-6-P by kidney mitochondria of either normal or aminonucleoside-nephrotic rats. These findings, and the additional observation of aminonucleoside-inhibition of kidney mitochondrial ATPase activity, appear to justify the conclusion that induction of aminonucleoside-nephrosis in the rat results in impaired kidney mitochondrial phosphorylation. Failure of aminonucleoside, added in vitro to normal rat kidney mitochondria, to significantly alter phosphorylation suggests that this effect is of secondary origin. Demonstration of inhibited phosphorylation when aminonucleoside is added in vitro to normal rat kidney submitochondrial phosphorylating complexes, however, suggests a primary effect. It is suggested that the mechanism of action of aminonucleoside may be bimodal, producing some of its metabolic effects either directly or indirectly through inhibition of reactions producing energy-rich ATP and some directly through inhibition of reactions by which the energy of ATP is released.

Glutathione metabolism in normal and induced states of growth.

Abstract 1. 1. Liver glutathione concentrations were found to be significantly higher in rapidly growing immature normal rats than in normal adult rats. Stimulation of the latter with growth hormone resulted in a significant elevation in liver glutathione concentrations. 2. 2. Stimulation of the growth process in the hypophysectomized immature rat with growth hormone resulted in a significant elevation in the concentration of liver glutathione. 3. 3. The percentage of administered 15 N found in the liver glutathione nitrogen obtained from normal adult rats and from hypophysectomized immature rats is markedly reduced as a result of stimulation of these animals with anterior pituitary growth hormone. This appears to be largely due to alterations in the size of metabolic pool of liver glutathione. The relatively low percentage of administered 15 N found in the nitrogen of liver glutathione obtained from rapidly growing immature normal rats compared with that obtained from normal adult rats, on the other hand, can be accounted for only partially in terms of the dilution effect of a larger metabolic pool of liver glutathione. 4. 4. The 15 N enrichment of the nitrogen of total liver proteins of rats in normal or induced states of growth, during a 2 hour period of assimilation of 15 N glycine, does not appear to be significantly different from that observed in rats which the growth process is arrested.

Respiratory control in kidney and liver mitochondria isolated from rats treated with the potent nephrotogenic aminonucleoside of puromycin

Abstract Rates of oxygen uptake of kidney mitochondria from normal and aminonucleoside-nephrotic rats have been determined by means of an oxygen electrode. The rates of oxygen uptake in state 3 (as defined by Chance and Williams) were lower for nephrotic rat kidney mitochondria than for normal kidney mitochondria. This change suggests that kidney mitochondrial enzymes from aminonucleoside-nephrotic rats may be decreased in amount, activity, or accessibility. It is also possible that mitochondria from aminonucleoside-nephrotic rats are less capable of maintaining structural integrity, and thus are damaged more during the isolation procedure. When livers of nephrotic rats were used as the source of mitochondria, impairment of respiratory control was not observed but, in fact, respiratory control ratios were higher than those of normal rat liver mitochondria. Respiratory rates were also obtained by preincubating normal kidney and liver mitochondria with aminonucleoside. No pronounced effect of aminonucleoside upon normal rat kidney mitochondria was observed in vitro .

Mechanism of Aminonucleoside-Induced Nephrosis in the Rat. IV. Hepatic Mitochondrial Oxidative Phosphorylation.

Summary 1. Phosphorylation associated with the oxidation of succinate is significantly higher in aminonucleoside-nephrotic rat liver mitochondria than in normal rat liver mitochondria. This alteration is induced as early as the 4th day during a 10-day period of induction of the disease with aminonucleo-side. 2. Phosphorylation associated with the oxidation of a-ketoglutarate, malate, fuma-rate, and β-hydroxybutyrate is not significantly altered in liver mitochondria of the aminonucleoside-nephrotic rat. 3. Phosphorylation associated with the oxidation of succinate is also increased in normal rat liver mitochondria incubated directly with aminonucleoside. In similar experiments conducted with rat liver submitochondrial phosphorylating complexes, however, aminonucleoside inhibits oxidative phosphorylation. We are indebted to Dr. J. M. Ruegsegger, Lederle Medical Research Section, American Cyanamid Co., for aminonucleoside used in this study.

Effects of buffer systems on the interaction of the potent nephrotogenic aminonucleoside of pur014ycin with rat kidney cortex microsomes

Abstract Study of the effects of pH on the interaction of the nephrotogenic aminonucleoside of puromycin with rat kidney cortex microsomes clearly indicates that binding of aminonucleoside to the microsomal fraction is sharply reduced when equilibrium dialysis experiments are conducted in the presence of the Good series of buffers, and in the presence of glycylglycine or phosphate buffers. Marked similarity in binding values, obtained in the presence of the Good series of buffers over the pH range 6.21 to 8.71, suggests a common mechanism of interference of these buffers with the binding of aminonucleoside to the microsomal fraction, and a buffer effect independent of pH. In experiments conducted with any one buffer compound, adjusted to different pH values, however, a pH effect was discernible.

Observations on rat kidney mitochondrial adenosine triphosphatase activity during induction of aminonucleoside nephrosis

Abstract 1. Kidney mitochondrial ATPase activity, assayed in the presence and absence of a variety of activating ion agents, has been determined at several time intervals during the course of induction of aminonucleoside disease in the rat. 2. In terms of cation transport, the most significant finding would seem to be an elevation in the multiplecation-activated (i.e. Mg ++ + Na + + K + ) kidney mitochondrial ATPase induced as early as the 4th day of treatment of rats with aminonucleoside. 3. Possible implication of multiple-cation-activated kidney mitochondrial ATPase in cation transport is discussed.

Mechanism of Aminonucleoside-Induced Nephrosis in the Rat. II. Metabolism of Aminonucleoside-8-C14.∗:

Summary Detailed studies were made of the chemical nature of carbon-14 activity found in acid-soluble nucleotide, ribonucleic acid, and deoxyribonucleic acid fractions isolated from kidney or liver tissue of rats rendered experimentally nephrotic with a series of 10 small daily injections of aminonucleoside-8-C14, and, in similar fractions obtained from rats sacrificed as early as 3 hours after administration of a single large dose of the labeled compound. No significant differences were noted. Only the adenosine and guanosine phosphates of the acid-soluble nucleotide fractions and the adenine and guanine bases of RNA and DNA hydrolysates were labeled with carbon-14. No evidence was obtained either for conversion of the intact aminonucleoside to the corresponding nucleotide or for incorporation of the 6-dimethylamino- or 6-methylamino-purine moieties into ribonucleic acid or deoxyribonucleic acid of kidney or liver tissue.

Mechanism of aminonucleoside-induced nephrosis in the rat. I. Metabolism of tritiated aminonucleoside.

Summary 1. The preparation and metabolism of radiochemically pure aminonucleoside, 6-dimethylamino-9-(3′-amino-3′-deoxy-β-D-ribofuranosyl)-purine, labeled with tritium by the Wilzbach procedure, is described and discussed. Data are presented for the distribution of radioactivity in body fluids and urine as well as in kidney and liver nucleic acids. 2. Study of the chemical nature of tritium activity in an acid-soluble nucleotide fraction, prepared from pooled kidney tissue of rats rendered nephrotic with the tritiated aminonucleoside, indicates that the rat is capable of metabolizing the latter into labeled products utilizable in the biosynthesis of adenosine and cytidine nucleotides. 3. Labeling of these nucleotides, which cannot be accounted for in terms of an exchange mechanism involving the hydrogens of body water, is considered in terms of alternative metabolic pathways.

Effects of the potent nephrotogenic aminonucleoside of puromycin on mitochondrial mechanochemistry

Abstract Neither spontaneous swelling nor U-factor formation is significantly altered in kidney mitochondria of rats treated for a period of 4 days with the nephrotogenic aminonucleoside of puromycin. Administration of the drug for an additional 6-day period, however, results in significant increases in initial swelling rates, extent of swelling, and level of U-factor in kidney mitochondria. Incubation of either normal rat kidney 01 liver mitochondria with 10 −4 M aminonucleoside results in enhancement of initial swelling rates, with the effect on liver mitochondria being considerably greater than that on kidney mitochondria. Aminonucleoside in vitro also enhances the initial rate of swelling induced in liver mitochondria with orthophosphate. Since little or no change occurs in U-factor formation during the initial period of orthophosphate-induced swelling, a mechanism of aminonucleoside enhancement of mitochondrial swelling that operates independently of U-factor formation may be assumed. Incubation of normal rat liver mitochondria in the presence of an equimolar concentration of adenine, aminonucleoside, or adenosine monophosphate results in a reduction in the time of onset of orthophosphate-induced swelling, this reduction being least with adenine and greatest with adenosine monophosphate.