Van R. Potter

Nuclei from Rat Liver: Isolation Method That Combines Purity with High Yield

A procedure is described that gives almost quantitative separation of clean nuclei from a homogenate of rat liver. It is a modification of methods that use concentrated sucrose solutions rather than citric acid or detergents and, therefore, permits further quantitative fractionation of cytoplasmic components.

Studies on free and membrane-bound ribosomes in rat liver: I. Distribution as related to total cellular RNA☆

The distribution of RNA in rat liver has been determined to be: 80% ribosomal RNA, 15% non-sedimentable RNA, and 4 to 5% nuclear RNA. Ribosomal RNA which occurs in free ribosomes, not attached to membranes, has been found to amount to 20% of the cellular RNA. Membrane-bound ribosomes contain 60% of the cellular RNA. Both the ribosomal and non-sedimentable RNA fractions have been characterized in terms of their sedimentation profiles in sucrose gradients. The amount of RNA per mg DNA in fed rats is increased over the amount found in fasted rats; however, the distribution of RNA among the fractions studied is not significantly different between fasted and fed rats. There are approximately 6 × 106 ribosomes per average liver cell in fed rats, with approximately 10 molecules of non-sedimentable RNA per ribosome.

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

Abstract A technique is described by which the intracellular water space can be determined in cells which are attached to a culture dish. A major advantage of this procedure is that the cells maintain their normal morphology. This technique takes advantage of the transport of the nonmetabolizable hexose, 3- O -methyl- d -glucose, to an intracellular concentration equal to the extracellular concentration, and the inhibition of 3- O -methyl- d -glucose efflux by phloretin. It requires only a determination of the nanomoles of hexose taken up at equilibrium plus a protein or cell number determination.

Enzyme inhibition in relation to chemotherapy.

Summary 1. The effect of enzyme concentration on the inhibition produced by certain inhibitors of the succinic dehydrogenase system has been experimentally determined. The inhibitors studied were malonate, itaconate, oxalacetate, quinone and cupric ion. 2. The reaction between succinic dehydrogenase and copper or quinone was not immediate but required 30 to 40 minutes when the amount of inhibitor was just sufficient to produce complete inhibition. 3. The effect of the strengths of binding of the inhibitor with the enzyme on the percent inhibition produced is shown to be related to the enzyme concentration. 4. An expression has been developed which relates the velocity of reaction to enzyme concentration in enzyme-inhibitor systems. 5. The results are discussed in relation to the chemotherapy of cancer. It is pointed out that the selective inhibition of an enzyme not unique to cancer tissue is theoretically possible.

Studies on free and membrane-bound ribosomes in rat liver: II. Interaction of ribosomes and membranes☆

Abstract Some evidence has been obtained that neither mRNA nor transfer RNA is the binding molecule between ribosomes and membranes. (1) In vivo treatment with actinomycin D, carbon tetrachloride or puromycin led to a conversion of heavy polysomes into light polysomes and single ribosomes without changing the distribution between free and bound ribosomes. (2) Amino acid incorporation in vitro converting polysomes into single ribosomes did not result in detachment of bound ribosomes from the membrane. (3) A 22% release of in vitro labeled peptidyl transfer RNA from membrane-bound ribosomes following puromycin treatment did not release bound ribosomes from the membrane. Ribosomes when bound to the membrane are resistant to pancreatic ribonuclease, whereas free ribosomes are converted into non-sedimentable products.

Distribution of radioactivity between the acid-soluble pool and the pools of RNA in the nuclear, nonsedimentable and ribosome fractions of rat liver after a single injection of labeled orotic acid

Abstract 1. 1. The distribution of acid-soluble and acid-insoluble radioactivity in homogenates of rat liver and the distribution of acid-insoluble radioactivity in various cell fractions of homogenates have been studied in a balance-sheet manner at various time points following a single intraperitoneal injection of labeled orotic acid. 2. 2. Equilibrium between the amounts of radioactivity in the acid-soluble pool and in the pool of nuclear RNA reached a ratio of about 8:1 by 3 h and remained constant from that time on. By the same criterion, equilibrium between the amounts of radioactivity in the acid-soluble pool and in the total acid-insoluble pool was reached after 36 h, with approx, 20% of the total radioactivity in the acid-soluble fraction. 3. 3. The apparent half lives of RNA in the nuclei, total ribosome fraction and nonsedimentable RNA were found to be 120 ± 13, 99 ± 11 and 90 ± 11 h, respectively. Similar half lives were calculated from the decay of the total radioactivity in these fractions per mg DNA. The total radioactivity per mg DNA in the acid-soluble fraction decayed with an apparent half life of 104 h. 4. 4. Evidence for reutilization of nucleotides has been obtained.

Isozyme studies on adult, regenerating, precancerous and developing liver in relation to findings in hepatomas

Studies on pyruvate kinase, hexokinase and aldolase isozymes were carried out over the first 4 months of feeding the carcinogen 3′-methyl-4-dimethyl-aminoazobenzene to rats. The observed changes in precancerous liver have been compared to the patterns observed in fetal, neonatal and regenerating liver and in hepatomas. Ingestion of the carcinogen over a period of time caused a marked loss of glucokinase and type-I pyruvate kinase and an increase in the isozymes characteristic of the fetal state. The largest increase in the fetal-type isozymes occurred during the period of rapid hyperplasia associated with the formation of hyperplastic nodules in the liver between the 2nd and 3rd months of carcinogen feeding. Primary large hepatomas induced by this diet were found to be characterized by high levels of the fetal-type isozymes. Studies on the changes in these isozymes during fetal and early neonatal development revealed that changes in the population of hemopoietic cells within the liver affect the isozyme patterns. The type-II pyruvate kinase and aldolase A appear to be located in the hemopoietic cells whilst type-III pyruvate kinase and aldolase B are located in the hepatocytes. The enzymes characteristic of the adult tissue, glucokinase and the type-I pyruvate kinase, are absent and present in low amounts, respectively, in neonatal rat liver. Changes in these isozymes following partial hepatectomy of adult rats suggest that when adult hepatocytes need to undergo cell division they cease to produce some of the isozymes characteristic of the fully differentiated state and revert to the production of isozymes characteristic of the fetal stage of liver ontogeny. Thus the appearance of fetal isozymes in precancerous liver is more probably due to increased hyperplasia rather than to the onset of malignancy. However, following controlled hyperplasia and differentiation after partial hepatectomy complete liver function is restored, whereas in the precancerous liver there is a loss of control of growth and differentiation in some cells resulting in hepatoma formation. The results have been discussed in terms of the concept of “Oncogeny as blocked Ontogeny” which offers a rational explanation of the diverse phenotypes observed in the transplantable Morris liver hepatomas. Particular attention has been focused on the significance of the appearance of the hyperplastic liver nodules in the process of carcinogenesis.

Food and light as separate entrainment signals for rat liver enzymes

Abstract The rat liver enzymes, tyrosine aminotransferase and ornithine decarboxylase, and to a lesser extent glucokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase, undergo diurnal oscillations when rats are entrained to a daily schedule of 12 hr of darkness and either 2 or 8 hr of access to food during the dark period. These enzymes increase in activity after feeding, but the patterns of activity versus time after feeding differ for rats with food available for 2 hr in the middle of the dark period, compared with the patterns for rats with food available for 2 or 8 hr at the beginning of the dark period. Therefore, induction of these enzymes involves interaction between dietary factors and hormonal factors related to light and dark periods. It appears that both the onset of darkness and the beginning of the light period are signals that interact with dietary and hormonal factors. While no single protocol can be optimal for all purposes, it is suggested that the 2 + 22 protocols in trained animals with other variables controlled may be more useful than other protocols, since in contrast to the 8 + 16 or ad libitum regimens, the animals receive their food during an interval of time that can be specified.

DNA Synthesis and Interaction between Controlled Feeding Schedules and Partial Hepatectomy in Rats

The rate of DNA synthesis has been measured during liver regeneration in rats adapted to a controlled feeding schedule. The results show two different phenomena in the regulation of DNA synthesis. The first is the appearance of a peak of DNA synthesis following the operation itself and independent of the time of the day; the second one is the presence of constant diurnal variations in the rate of DNA synthesis in response to the partial hepatectomy and following the stimulus or stimuli of the controlled feeding schedule.

Enzyme levels in rats adapted to 36-hour fasting☆

Abstract In an attempt to increase the amplitude of the normal daily oscillations in nucleic acid metabolism and enzyme synthesis or activation, male rats were adapted to a feeding program in which food was available for 12 hr in each 48-hr period, with lighting automatically regulated to give alternating 12-hr periods of light and darkness. Thus food was available only during alternate 12-hr periods of darkness. Controls were fed on an ad libitum basis. The “36-hr fasting-adapted” animals showed great physical activity on the nights they were not fed, and when kept in exercise cages traveled an average of 15.3 km per 12-hr period of fasting. The fasting-adapted animals weighed much less than the ad libitum controls, and after 30 days weighed an average of 225 g in contrast to 325 g when both groups began the experiment at 150 g body weight. Glycogen, blood sugar, the activities of glucokinase, hexokinase, glucose-6-phosphate dehydrogenase, citrate cleavage enzyme, tryptophan pyrrolase, tyrosine transaminase, serine deaminase, and ornithine transaminase, and labeling of RNA and DNA in vivo were measured. Several of the enzymes showed great increases in the amplitude of cyclic fluctuations in activity. Some enzymes showed evidence of a parallel fluctuation, while others showed individualized timing in peak activity. The 36-hr fasting-adapted animal may be useful in studies on mechanisms of regulating enzyme synthesis or activation and may suggest how human subjects might undertake a program of stress-conditioning.