Double Isotope Technique Research Articles

On the Measurement of Protein Turnover in Animal Cells

The double isotope technique of Arias, Doyle, and Schimke ((1969) J. Biol. Chem. 244, 3303) with radioactive leucine as precursor has been modified to yield accurate values for rate constants of protein degradation. The advantages of the method are that it is both rapid and reproducible in that the protein whose half-life is being measured is isolated only once from one experimental organism and that the values obtained for the rate constants of degradation are comparable to those measured with [guanidino-14C]arginine, and that unlike the latter precursor, radioactive leucine is extensively incorporated into liver protein. The turnover of the population of total soluble proteins and six proteins isolated in homogeneous form from rat liver was studied by means of the double isotope method. The majority of liver proteins are degraded with half-lives between 2 and 8 days. There is a general correlation between subunit size and rate of degradation. The subunits of the multimeric proteins δ-aminolevulinate dehydratase, catalase, ferritin, and lactate dehydrogenase are degraded at the same rate as the protein in its quaternary configuration. No intermediate states in the degradation of the subunits were found in the biologically active proteins.

The relation between cholesterol absorption and cholesterol synthesis in the baboon.

To determine the relation between cholesterol absorption, total endogenous cholesterol synthesis, and hepatic cholesterol synthesis in a primate, cholesterol synthesis has been studied in biopsies of liver and ileum from normal baboons fed varying amounts of cholesterol and in biopsies of liver from baboons that had been subjected to ileal diversion. In addition, total cholesterol production rates, cholesterol absorption, and total endogenous cholesterol synthesis have been measured in these animals by a double isotope technique in which the animals were given a single injection of cholesterol-4-(14)C and fed constant amounts of cholesterol-1,2-(3)H for 4 months. From these studies, it has been concluded that on a low cholesterol intake cholesterol synthesis in the liver accounts for about three-fourths of total endogenous cholesterol production. The feeding of cholesterol produces complete inhibition of hepatic synthesis in the normal animal only when absorption approximates the amount synthesized by the liver when no cholesterol is fed, e.g., 400-500 mg/day. Finally, the intestine, which does not possess complete negative feedback control of cholesterol synthesis when cholesterol is fed, may be a significant site of nonhepatic cholesterol synthesis in these animals. In studies of four baboons subjected to ileal diversion, it was concluded that the regulation of cholesterol synthesis is distinctly different when the enterohepatic circulation is interrupted. These animals did not exhibit negative feedback of hepatic cholesterol synthesis when cholesterol was fed, despite the fact that cholesterol absorption approximated that of normal animals fed similar diets. The inference has been drawn that bile acids may be involved directly or indirectly in the regulation of hepatic cholesterol synthesis in this species or that the ileum itself may modulate the hepatic negative feedback.

Studies on thyroidal colloid pinocytosis using a double isotope technique.

ABSTRACT The process of secretion of the thyroid hormone involves several steps: pinocytosis of thyroglobulin, fusion of the colloid droplets with the lysosomes, digestion of thyroglobulin by a cathepsin, dehalogenation of tyrosines and release of thyronines into the blood stream. The present paper describes a double isotope technique for studying the first two steps. Thyrotrophin (TSH) administration to rats increased the radioactivity present in all fractions, specially in the 15 000 × g pellet. When the subcellular distribution of acid phosphatase was determined, the highest specific activity was found in this fraction, thus indicating the presence of lysosomes. The content of radioactive materials in the 15 000 × g pellet was analyzed by trichloroacetic acid precipitation and by ascending paper chromatography using n-butanol:ethanol:ammonium hydroxide (5:1:2;v/v) as solvent system. The results obtained showed that 90% of the radioactivity was protein bound and strongly suggest that this material is thyroglobulin.

Synthesis of Nuclear-Associated Proteins by Lymphocytes Within Minutes After Contact with Phytohemagglutinin

Abstract The interaction of lymphoid cells with phytohemagglutinin (PHA) gives rise to a series of chemical and morphologic changes that culminate in the mitosis of the lymphoid cell (1). Although mitosis is not detectable for 2 to 3 days after contact with PHA, earlier changes in the lymphocyte have been described (2–17). The molecular mechanism underlying the activation of the lymphocyte are unknown and little is known concerning the chemical nature of the proteins synthesized shortly after contact with PHA. Using a double isotope technique we have identified a class of nuclear proteins whose synthesis is stimulated within 20 min after contact with PHA. These proteins may be associated with nucleic acids and represent aggregates with a molecular weight greater than 1,000,000. Lymph nodes were obtained from strain 2 guinea pigs 2 to 6 weeks after injection with 0.1 ml of complete Freund's adjuvant into each of the foot pads.

Turnover and Exchange of Ribosomal Proteins from Rat Liver

Abstract Rat liver ribosomes are not synthesized and degraded as functional units but are in a continual state of flux in which most protein constituents of the ribosome rapidly exchange with similar ribosomal proteins in the cytoplasm. This conclusion is based on two main types of evidence. 1. The relative degradation rates of ribosomal proteins are heterogeneous. The relative rates of degradation of ribosomal proteins were studied by a double isotope technique in which a single injection of [14C]leucine was administered to rats 5 days prior to [3H]leucine. As indicated by 3H:14C ratios of ribosomal proteins separated on sodium dodecyl sulfate-acrylamide gels, there is a marked heterogeneity of turnover of ribosomal proteins and a general correlation between relative degradation rates and the molecular size of the protein. This general relationship was also found for soluble proteins (Dehlinger, P. J., and Schimke, R. T. (1970) Biochem. Biophys. Res. Commun., 40, 1473). One possible explanation for this similarity is that protein constituents of ribosomes are degraded primarily as free cytoplasmic proteins. 2. Ribosomal proteins in the cytoplasm appear to be in equilibrium with the protein components of ribosomes since most protein constituents of ribosomes easily exchange in vitro with cytoplasmic proteins. This exchange was demonstrated by adding radioactive ribosomes to a nonradioactive supernatant fraction, or vice versa, and comparing the specific activity of the ribosomes before and after exchange. Two classes of exchangeable proteins can be identified. At least 70% of the ribosomal protein exchanges rapidly at 8°, while an additional 20% exchanges at 37°. The protein constituents of polysomes, monosomes, and subunits and both split and core proteins, prepared by washing ribosomes with 0.4 m KCl, are involved in the exchange process. The exchanged proteins have mobilities in sodium dodecyl sulfateacrylamide gels which are similar to those for proteins originally present on ribosomes. Free ribosomal proteins may constitute as much as 17% of the total soluble protein, indicating that in the rat liver there are, on the average, 10 times as many ribosomal proteins in the soluble form as are constituents of ribosomes.

Studies on steroid conjugates. X. Significance of conjugation involved in biosynthesis of 2-methoxyestrogen.

The role of the conjugate in the biosynthesis of 2-methoxyestrogen in the living animal has been explored by the double-isotope technique. The isotope ratios of the isomeric catechol estrogen monomethyl ethers excreted in urine after simultaneous administration of estrone-4-14C and estrone-6, 7-3H sulfate or estradiol-6, 7-3H 3-glucuronide were determined. Comparison of the 3H/14C value with that of the administered steroid implies that the sulfate conjugation may possibly participate in the predominant formation of 2-methoxyestrogen, while the conjugation with glucuronic acid may not. The dimethyl ether of catechol estrogen was metabolized with random O-demethylation yielding two isomeric monomethyl ethers both in the rat and man. Therefore it appears that the selective O-demethylation may not be involved in the preferential formation of 2-methoxyestrogen.

Plasma catecholamines in hypertension.

Plasma catecholamines were measured by employing a precise and sensitive double-isotope technique in 9 control subjects and in 20 patients with various types of hypertension. Venous blood was collected while the subjects rested in the supine position. In the control subjects total plasma catecholamine concentration averaged 0.25 ng/ml ± 0.08. The noradrenaline concentration was 0.21 ng/ml and the adrenaline concentration 0.05 ng/ml. Total plasma catecholamine concentration was normal in patients with renal and essential hypertension while the 4 patients with phaeocromocytoma showed values ranging from 0.48 to 7.90 ng/ml.

Combined pancreas and liver scanning using a double isotope technique.

Abstract Experience has been gained in using the dual radioisotope method routinely to scan the pancreas in over 200 patients. Results are presented for the first 129 patients for both the pancreas scans and the gamma-camera scintigrams taken at the same time. These show that a normal pancreas scan virtually excludes disease of that organ. Three lesions were missed in a total of 38 patients. Abnormal scans showed a variety of presentations with no uptake or reduced uptake being a more common appearance. Space-occupying lesions may be defined, but the poor statistics of the method makes diagnosis difficult. In this selected series abnormal scans were nearly always associated with pancreatic disease or disease elsewhere. Sixty-two scans showed an abnormal appearance. Liver scintigrams were carried out on a gamma-camera before the pancreas examination. Of 52 normal scintigrams 46 were proved to be normal (87 per cent) while six space-occupying lesions were missed. Twenty out of 24 patients (83 per cent) had ...

Size Distribution of Membrane Proteins of Rat Liver and Their Relative Rates of Degradation

Abstract The proteins associated with various membrane fractions, including smooth and rough endoplasmic reticulum, plasma membrane, and mitochondria of rat liver, were solubilized with sodium lauryl sulfate and separated by electrophoresis on sodium lauryl sulfate acrylamide gels. Smooth and rough endoplasmic reticulum have similar electrophoretic patterns. All other fractions have characteristic patterns in which no single or predominant protein occurs. The smooth endoplasmic reticulum fraction was fractionated by use of Triton X-100, which solubilized approximately 50% of the membrane proteins. The patterns of Triton X-100-soluble and -insoluble proteins are distinctive. The relative rates of degradation of the proteins were studied by use of a double isotope technique in which 14C-leucine was administered to intact rats 4 days prior to administration of 3H-leucine. As indicated by the differences in 3H:14C ratios, there is a marked heterogeneity of turnover of proteins of both the endoplasmic reticulum and plasma membrane. There is a general correlation between the relative degradation rates of membrane proteins and the molecular size of the protein (subunit) as separated by electrophoresis in sodium lauryl sulfate acrylamide gels. This general relationship was found also for the soluble protein fraction of rat liver, whether fractionated by sodium lauryl sulfate acrylamide gels, or by Sephadex G-200 columns in the presence of sodium lauryl sulfate. A model in which membrane proteins associated and dissociate from the protein-lipid complex is favored to explain the heterogeneity of degradation, and the general correlation of size with degradation rate.

Effect of age, sex, starvation, hypophysectomy and growth hormone from several species on the inorganic sulphate pool and on the incorporation in vivo of sulphate into mouse costal cartilage. An attempt to study sulphation factor activity in vivo.

ABSTRACT The influence of several conditions on sulphate incorporation into costal cartilage was investigated in mice of the N.M.R.I.-strain and in dwarfmice (dw/dw) by a new double isotope technique. The method permits estimation of sulphate incorporation into costal cartilage in vivo after correction for the size of the exchangeable inorganic sulphate pool of the individual animal. Detoxication to phenyl sulphate of injected phenol, following an injection of 3H-phenol and 35S-sulphate into the mice, allows determination of the sulphate pool. The incorporation rate of sulphate is expressed in ng, and includes all SO4 which had been incorporated in vivo into 5 pairs of cartilaginous ribs including their osteochondral junctions during 30 minutes. Sulphation rate was rather constant in growing female mice between 3 and 8 weeks age and then decreased markedly. Growing male mice exhibited an increase of sulphation activity until an age of 5 weeks and then a gradual decrease, similar to that of the females. Growth rates of both sexes were positively correlated with sulphate uptake activities. The form of the disappearance curve of incorporated sulphate suggested that the standard cartilage sample includes regions with different sulphate-metabolizing activities. Hypophysectomy resulted in a decrease of sulphation rate to a level about 30% of the control values, which was constant for several weeks. Starvation of the animals likewise depressed the sulphation rate and after 72 hours starvation the values became similar to those after hypophysectomy. Dwarf mice had much lower sulphation activities than their heterozygous controls. Neither bovine GH nor human GH elevated the low sulphation activity of hypophysectomized mice but porcine and ovine GH-preparations had a pronounced and dose-dependent effect. Normal growing male and female mice showed no response to large doses of ovine GH. Ear, xiphoid and tracheal cartilage from hypophysectomized animals behaved as costal cartilage with respect to the reduction in sulphation rate, but GH restored the sulphation values to normal only in costal cartilage. A single dose of 1 mg ovine GH given to hypophysectomized mice at different times in relation to the sulphation experiment increased sulphate incorporation only after a time lag of more than 8 but less than 17 hours after injection of GH, whose effect lasted at least 38 hours.

�ber den Einflu� einer befristeten Unterbrechung der Nebennierendurchblutung auf die Steroidbiosynthese und die ACTH-Steuerung der Nebennierenrinde

The effect of general damage to suprarenal gland cells on the bio-synthetic output of steroid hormones by the adrenal cortex was examined in rabbits and compared with the state of the energy metabolism. For this purpose the suprarenal glands were cut off from their blood supply for up to 120 min. Regarding the chief steroid of the suprarenal gland, corticosterone, the secretion at maximum ACTH stimulation was 17.89 nmol/min; this level dropped to 5.83 nmol/min after 60 min of ischemia and 30 min of recovery, and to 2.38 nmol/min after 120 min of ischemia and 30 min of recovery. A comparison of these findings with the other data revealed a disturbance of steroid biosynthesis in the form of an inhibition of 11β hydroxylation. Maximum ACTH efficiency at ACTH saturation is proportional to the time of damage; it can be expressed by the exponential equation ACTH-eff (max.)=17.9×exp (−0.0156×t) The meaning of the findings with regard to the mechanism of ACTH activity is discussed. In addition, possibilities are shown for the enzymatic conversion of steroids which cannot be acetylated in their primary form to compounds which can be determined by the double isotope technique.

Two protein fractions obtained from hepatic plasma membranes. Studies of their composition and differential turnover

1. 1. Plasma membranes were isolated from rat liver and their enzymatic, morphologic, and chemical composition was studied. Two protein fractions were isolated from a hepatic plasma membrane preparation using either 33% aqueous pyridine or 0.05 M K 2CO 3. One was water-soluble, contained 50% of the original membrane protein; the other was water-insoluble, contained the remaining protein and the majority of lipids. With 0.5 M K 2CO 3 the majority of sialic acid was in the water-insoluble fraction. Neutral carbohydrates were equally distributed between the two fractions. 2. 2. Polyacrylamide gel electrophoresis of water-soluble fractions in sodium dodecyl sulfate revealed 15–20 protein bands which were also present in the intact plasma membrane. 3. 3. Relative protein turnover was determined by a double isotope technique. The water-insoluble fraction had a greater turnover ratio than the water-insoluble fraction. These studies are consistent with a “multistep process” of plasma membrane synthesis.

" FREE " IRON LOSS IN ATROPHIC GASTRITIS, POST-GASTRECTOMY STATES, AND ADULT CŒLIAC DISEASE

A double isotope technique has been used to measure separately the blood and non-hæmoglobin iron loss from the gastrointestinal tract in iron deficiency. Groups of patients were examined who had had a partial gastrectomy, or who had atrophic gastritis or adult cœliac disease. Preliminary results show increased " free " iron loss in all three conditions.

Simultaneous clinical measurement of regional blood flow and effective capillary surface in the human skeletal muscle by a double-isotope technique: Observations on the effect of locally injected norepinephrine

A double-tracer method using a water-soluble (Na-24) and a lipid-soluble highly diffusible substance (Xe-133), and measuring the local tissue clearance of both substances simultaneously, has been applied to the study of regional blood flow and transport function in normal and subnormal perfusion states in human skeletal muscle. As model situations resting muscle with and without the local application of norepinephrine have been investigated. Locally injected norepinephrine reduces both capillary blood flow and capillary surface area. At the concentrations used the latter was reduced to one third the value in resting skeletal muscle. It is concluded that blood flow is not a sufficient parameter for the evaluation of transport of water-soluble substances to and from tissue. Equal capillary flow can give quite different transcapillary exchange, giving a clearance of water-soluble substances between 100 and a few per cent of capillary blood flow in the flow range of resting skeletal muscle, depending on available capillary surface. The method outlined gives a means of identifying the actual state with regard to this. A theoretical background to the method is given and some of the difficulties of the approach are discussed.

A single method for determination of urine steroids*I. Levels of testosterone, epitestosterone and 17-ketosteroid fractions, and II. Specific activities of testosterone, androsterone and etiocholanolone and production rate of testosterone following administration of labelled testosterone

Summary o 1. A method for the measurement of urinary testosterone, epitestosterone and 17-ketosteroid fractions has been established. The testosterone and epitestosterone are assayed by the double-isotope technique, utilizing 14 C-acetic anhydride for quantitation and the addition of 3 H-testosterone glucuronoside and 3 H-epitestosterone to correct for losses during the procedure. The 17-ketosteroids are separated on a gradient elution alumina column and measured with the Zimmermann reaction. 2. The method can be used to isolate urine metabolites of administered 3 H-testosterone; the specific activity of the isolated urine testosterone can be used to calculate a testosterone production rate in men. The specific activities of urine testosterone, androsterone and eticholanolone and the testosterone production rate prior to and during estrogen administration are reported for two men.

A single method for determination of urine steroidsI. Levels of testosterone, epitestosterone and 17-ketosteroid fractions, and II. Specific activities of testosterone, androsterone and etiochloanolone and production rate of testosterone following administration of labelled testosterone

Summary o 1. A method for the measurement of urinary testosterone, epitestosterone and 17-ketosteroid fractions has been established. The testosterone and epitestosterone are assayed by the double-isotope technique, utilizing 14 C-acetic anhydride for quantitation and the addition of 3 H-testosterone glucuronoside and 3 H-epitestosterone to correct for losses during the procedure. The 17-ketosteroids are separated on a gradient elution alumina column and measured with the Zimmermann reaction. 2. The method can be used to isolate urine metabolites of administered 3 H-testosterone; the specific activity of the isolated urine testosterone can be used to calculate a testosterone production rate in men. The specific activities of urine testosterone, androsterone and eticholanolone and the testosterone production rate prior to and during estrogen administration are reported for two men.

Iron absorption in chronic renal disease.

1. Absorption of inorganic iron was studied in thirty-four patients with chronic renal failure by a double isotope technique. 2. Eight patients with normal iron balance had a mean absorption of 3·5%, ten patients with iron overload had a mean absorption of 3·6%, and sixteen patients with iron depletion had a mean absorption of 58%. Thus, alterations in absorption appeared to be related to disturbances in iron balance. 3. The rate of erythropoiesis had no evident effect on iron absorption nor did the degree of anaemia. 4. The presence of renal disease and the degree of azotaemia likewise did not appear to affect absorption.

In vivo behaviour of frozen and freeze-dried fibrinogen and of that prepared from out-dated blood

1. (1) The effect of freezing and freeze-drying on the metabolic behaviour of fibrinogen has been investigated by double isotope techniques using a differently labelled native aliquot from the corresponding batches of fibrinogen as a control. The results indicated that purified human or rabbit fibrinogen can be frozen and refrozen repeatedly without altering their biological properties. Freezing did not induce aggregation or partial polymerisation and frozen-dried rabbit fibrinogen behaved in vivo indistinguishably from the native protein. 2. (2) Human fibrinogen prepared from a pool of out-dated but properly stored blood was catabolised at the same rate as a simultaneously injected fresh control fibrinogen, except for a small fraction which was obviously denatured. 3. (3) Rabbit, monkey and human fibrinogens appeared to retain their metabolic integrity for at least several weeks when stored frozen at −25°. 4. (4) From a metabolic point of view it is concluded that advance preparations of fibrinogen for establishing frozen pools are permissible. However, to obtain results in vivo beyond doubt, such pools should be run on fibrinogen originating from fresh plasma.

Intestinal vitamin B12 absorption in the dog

Summary The aim of this study was to determine whether the intestinal absorption of vitamin B 12 in the dog is mediated by intrinsic factor. Dog gastric juice and duodenal mucosal extracts were tested for their ability to promote intestinal absorption of radioactive vitamin B 12 in vitro on guinea pig and dog intestinal mucosa homogenates and everted dog jejunal and ileal sacs. The absorption by in situ dog ileal sacs of radio B 12 alone and radio-B 12 bound to dog gastric juice or duodenal mucosal extracts into blood and liver was further studied in vivo by means of a double label isotope technique. In all systems the absorption of B 12 alone was higher than that of B 12 bound to dog gastric juice or duodenal mucosal extracts, which rather inhibited B 12 absorption. These studies do not support the notion that dog gastric juice and duodenal mucosal extracts are endowed with intrinsic factor activity and that, in the dog, physiological B 12 absorption in the intestine is mediated by intrinsic factor.

Kinetic analysis of plasma radioactivity after oral ingestion of radiocalcium.

THE absorption of calcium from the gastro-intestinal tract is one of the chief regulators of calcium metabolism and is known to be disturbed in a variety of clinical disorders. Despite its importance, however, no rapid, accurate procedure is available for its measurement, which normally requires the estimation of dietary and faecal calcium over a period of weeks. Several workers have therefore sought to establish an isotopic procedure which would enable calcium absorption to be calculated from the plasma radioactivity1,2 or residual faecal activity3,4 following an oral dose of radioactive calcium. It has been found, however, that although plasma radioactivity is inversely related to faecal recovery, it is not, by itself, a reliable measure of calcium absorption3,4. This is not surprising in view of the fact that the concentration of tracer at any given time after administration must be determined not only by the rate of its absorption into plasma, but also by the rate of its removal, and some workers have used double isotope techniques to overcome this difficulty5.