Antimycin A: stimulation of cell division and protein synthesis in Tetrahymena pyriformis.

Addition of antimycin A to a culture of Tetrahymena pyriformis caused an increase in cell division and protein synthesis in this ciliated protozoan. The antimycin effect is a function of the time of exposure to the antibiotic as well as of the age of the culture. A large accumulation of endoplasmic reticulum, reflecting increased protein synthesis, was visualized by electron microscopy in cells stimulated by the antimycin A.

Energy substrate utilization in freshly isolated Morris Hepatoma 7777 cells.

Freshly isolated Morris Hepatoma 7777 cells were prepared for a study of the utilization of palmitate and beta-hydroxybutyrate as metabolic fuels compared to other major energy substrates (glucose and glutamine). These cells were capable of oxidizing both [U-14C]palmitate and beta-[3-14C]hydroxybutyrate although the rates accounted for only 10 +/- 3 (SD) and 9 +/- 4% of total oxygen consumed, respectively; n = 4. Incorporation of [U-14C]glutamine and [U-14C]glucose carbon into 14CO2 made up for 38 +/- 13 and 9 +/- 2% of oxygen consumed by these cells, respectively. The conversion of glucose carbon into lactate was estimated to supply 26 +/- 6% of ATP. Thus, glutamine oxidation and lactate formation from glucose were the major contributors to estimated ATP needs. Conversion of these substrates into lipids was also studied and compared with oxidized products. Incorporation of glucose, glutamine, and beta-hydroxybutyrate into nonsaponifiable lipids and fatty acids was only 6.0 +/- 2.9, 0.8 +/- 0.2 and 17.7 +/- 6.65% (n = 3) of their respective rates of CO2 formation. This suggests that in freshly isolated Morris Hepatoma 7777 cells, citrate export from the mitochondria for cholesterogenesis and lipogenesis is a minor fate of substrate carbon entering the mitochondria for oxidation.

Substrate inhibition of carnitine palmitoyltransferase by palmitoyl-CoA and activation by phospholipids and proteins.

When carnitine palmitoyltransferase is purified it shows increasing substrate inhibition by palmitoyl-CoA as the protein content of the assay mixture is decreased. The purified enzyme is stimulated by addition of phospholipids (phosphatidylcholine, cardiolipin) and proteins (albumin, fatty acid-binding protein, lambda-globulin) to the reaction mixture. The effects of phospholipid and protein are more than additive, particularly with relatively high concentrations of palmitoyl-CoA. It is suggested that the enzyme contains hydrophobic sites which require phospholipid to prevent spurious binding of palmitoyl-CoA and which normally anchor the enzyme to the mitochondrial membrane.

Growth-dependent factors in the regulation of aminoacyl-tRNA synthetase activities of Tetrahymena pyriformis.

Changes in phenylalanyl-tRNA synthetase (L-phenylalanine : tRNAPhe ligase, EC 6.1.1.20) and leucyl-tRNA synthetase (L-leucine : tRNALeu ligase. EC 6.1.1.4) activities were studied during the growth cycle of Tetrahymena pyriformis. High levels of charged tRNA observed during exponential growth were associated with elevated aminoacyl-tRNA synthetase activities. Low levels of charges tRNA in the stationary phase culture were associated with decreased aminoacyl-tRNA synthethase activities together with a concomitant accumulation of factor(s) which inhibited the enzyme activities. The inhibitory factor(s) has been partially purified and evidence is presented to rule out RNA, RNAases, proteases and ATPases as the responsible inhibitory factor(s) of the aminoacyl-tRNA synthetases.

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation.

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328-334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8 degrees C for 3-4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25-30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66-79). At 0-4 and 10 degrees C, the Vmax and Km of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The Ki values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37 degrees C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in Vmax and a 20% decrease in Km for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA.

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.

Activity of long chain acyl-CoA synthetase in isolated alveolar type II cells.

Fatty-acyl-CoA synthetase activity was determined in rat alveolar type II cells. Compared to whole-lung homogenate, the enzyme specific activity with palmitic acid was 3.6-fold higher in isolated type II alveolar cells. The enzyme in rat alveolar type II cells did not discriminate among various fatty acids, suggesting that supply of fatty acids rather than specificity might be an important factor for their activation in these cells.

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.

Characterization and purification of fatty acid-binding protein in rat and human adipose tissue.

A protein with properties similar to fatty acid-binding protein has been isolated from rat and human adipose tissue. Comparison of fatty acid-binding protein from rat liver and adipose tissue and human adipose tissue shows that all have approximately similar molecular weights. Immunologically, rat liver fatty acid-binding protein is similar to the protein characterized from rat adipose tissue. In isolated rat fat cells the fatty acid-binding protein was demonstrated to be involved in the uptake and esterification of long-chain fatty acids. These observations constitute evidence for a potential role of this protein in the fatty acid metabolism of adipocytes.

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts.

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.

Comparison of the adenine nucleotide translocase in hepatomas and rat liver mitochondria.

Various biochemical properties of the adenine nucleotide translocase were compared with mitochondria prepared from control and host liver, and Morris hepatomas 7777, 7800 and 5123C. The transport of phosphoenolpyruvate on the adenine nucleotide translocase was found to be three to four times more active, and inhibition of the transporter by palmitoyl-CoA and atractylate considerably less in hepatoma the active transport of phosphoenolypyruvate was associated with a greater stimulation of calcium egress from the mitochondria matrix by the anion in the hepatoma. The diminished sensitivity of the adenine nucleotide translocase to palmitoyl-CoA in hepatoma mitochondria was associated with lower levels of long chain acyl-CoA esters in the whole tissue. A change in activation energy at 6 degrees C for the adenine nucleotide translocase was found in host liver mitochondria while no break point in the temperature curve was observed in hepatoma mitochondria. These results are most consistent with a change in the structure-function relationship of hepatoma mitochondria due to differences in lipid composition.

Rat islet mitochondrial adenine nucleotide translocase and the regulation of insulin secretion.

Employing a preparation of rat islet mitochondria, phosphoenolpyruvate has been shown to interact with the mitochondrial adenine nucleotide translocase. Thus, phosphoenolpyruvate inhibited mitochondrial uptake of [14C]ADP and exchanged with intramitochondrial [14C]ATP. A concentration-dependent inhibition of islet mitochondrial 45Ca2+ accumulation was seen when mitochondria were exposed to phosphoenolpyruvate with half-maximal inhibition observed at a phosphoenolpyruvate concentration of 0.2 mM. In experiments employing whole islets, phosphoenolpyruvate content was shown to be significantly elevated at both 1 and 30 min after an increase in the medium glucose concentration from 2 to 20 mM. In these experiments, the estimated islet concentrations of phosphoenolpyruvate fell in the range of maximal sensitivity of the islet adenine nucleotide translocase to phosphoenolpyruvate-induced inhibition of Ca2+ accumulation. It is concluded that increased concentrations of islet phosphoenolpyruvate resulting from increased extracellular glucose concentration may act to trigger or promote glucose-stimulated insulin secretion by modifying the distribution of Ca2+ between the islet cytosolic and mitochondrial compartments in a transport reaction catalyzed by the adenine nucleotide translocase.

Synergistic effects of male sex and obesity on hepatic insulin dynamics in SHR/Mcc-cp rat.

The effects of obesity and sex on hepatic insulin metabolism were evaluated in the SHR/Mcc-cp rat. During in situ liver perfusion, insulin clearance rate (CLR) expressed per gram of liver tissue was reduced by 58 and 68% in obese females and males, respectively, compared with lean controls. Male sex resulted in CLR reductions of 46% in lean and 59% in obese animals. Obesity resulted in 50% reduction of insulin-receptor binding to isolated hepatocytes. In both lean and obese animals, male sex also resulted in a decrease of approximately 34% in insulin binding. Scatchard plots indicated that the reduction in insulin binding was primarily due to a decrease in number of cell surface receptors. Receptor-mediated insulin degradation was 40% less in obese than lean animals. Male sex also resulted in 27% less insulin degradation relative to females. Receptor-mediated insulin partitioning between four compartments (cell surface bound, internalized and/or cryptic, degraded, and dissociated or released intact), expressed as a percentage of the initial membrane-bound hormone, did not differ between the animal groups. Thus, male sex and obesity are independently and additively associated with a reduction in hepatic insulin clearance and a decrease in the number of cell surface insulin receptors with a proportional decrease insulin compartmentalization and degradation. This mechanism may partly account for the synergistic effects of male sex and obesity on the degree of hyperinsulinemia and insulin resistance and the predisposition to diabetes.

Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA.

A long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Carboxyatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not carboxyatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins.

The carbon pathway for lipogenesis in isolated adipocytes from rat, guinea pig, and human adipose tissue.

The synthesis of fatty acids from a variety of labeled substrates by isolated adipocytes of the rat, guinea pig, and human was investigated. The incorporation of radioactive glucose and pyruvate into triglyceride fatty acids was considerably lower in human than either rat or guinea pig adipose tissue. By contrast, the incorporation of palmitate into adipose tissue triglycerides was approximately the same in all three species. End carbon analysis of fatty acids isolated from adipocytes incubated with pyruvate-U-14C indicated that although the synthesis of fatty acids in human adipose tissue was extremely low compared to that of the rat and guinea pig, it represented de novo biosynthesis rather than chain elongation of existing fatty acids. It is suggested that in the human, fatty acids are synthetised de novo primarily in the liver. In adipose tissue, lipogenesis consists essentially of the esterification of fatty acids, obtained from plasma, into triglycerides.

Perturbation of serum carnitine levels in human adults by chronic renal disease and dialysis therapy.

Serum carnitine levels in nondialyzed and dialyzed patients with chronic renal disease were compared against a group of normal control subjects. The concentration of serum carnitine was directly correlated with that of serum creatinine (r = +0.734; p less than 0.001). In nondialyzed uremic patients the serum free carnitine levels in males rose 218% (p less than 0.001) and in females rose 186% (p less than 0.001) above normal control values. During dialysis there was a sharp decline in serum carnitine to levels reaching 20% of the zero time control value (p less than 0.001). The decrease in serum carnitine could be accounted for by an almost quantitatively accumulation of carnitine in the dialysate fluid. After termination of dialysis there was a hyperbolic rise in serum carnitine which reached the high values again within 44 to 48 h. It is postulated that frequent perturbations in serum carnitine as a result of chronic dialysis therapy over a prolonged time period could potentially lead to a tissue deficiency in carnitine with its resultant complications.

Host and tumor growth and energy substrates in blood of hepatoma-bearing rats receiving high-fat parenteral infusions.

We examined the effects of isocaloric carbohydrate-based TPN vs fat-based TPN on plasma levels of energy substrates and insulin and on growth of Morris Hepatoma 7777 and host animals. Hepatoma-bearing rats fed similar diets ad libitum served as controls. Although no differences in tumor growth were observed, host weight loss was less in parenterally than in orally fed animals. Liver lipid and plasma free fatty acids and triglycerides in tumor-bearing and normal rats infused high-fat TPN were markedly higher than in all other groups, suggesting that this level of lipid infusion exceeds the rate of utilization. Plasma glucose and insulin in tumor-bearing rats infused high-fat TPN were higher than in all other groups, which may indicate insulin resistance. Replacing glucose energy in TPN with lipid does not appear to reduce glucose availability to tumors but does have potentially deleterious effects on the host.

Dietary carnitine intake related to skeletal muscle and plasma carnitine concentrations in adult men and women.

The purpose of this investigation was to determine if there was any relationship between dietary carnitine intake and the concentrations of carnitine in skeletal muscle and blood plasma in healthy adult men and women. Subjects (14 men, 14 women, fasted 8 h) reported to the Biodynamics Laboratory where they completed a 24-h diet recall questionnaire. Resting muscle biopsy (vastus lateralis) and blood plasma samples were taken and assayed for free, short-chain, and long-chain acyl carnitine concentrations. Dietary carnitine intake was estimated from data on concentrations in food. There was no significant relationship between either protein or carnitine intake with skeletal muscle carnitine concentrations. There was a significant relationship between both dietary carnitine (r = 0.50) and protein (r = 0.48) intake with blood plasma total acid soluble carnitine concentrations (p less than 0.01) in all subjects.

Effect of oral alanine loads on the serum triglycerides of oral contraceptive users and normal subjects.

The effect of orally administered L-alanine loads on serum triglycerides, and plasma insulin and glucose, was studied in 23 women using an estrogen-containing oral contraceptive and 13 healthy female controls. Oral contraceptive users had significantly higher fasting serum triglycerides than the controls. Serum triglycerides concentrations udnerwent little changes in the controls after alanine ingestion, whereas the oral contraceptive users showed increases which were maintained throughout the 3-hr sampling period. The two groups had similar elevations in plasma insulin after alanine loading; the glucose concentrations were unchanged. The changes in serum triglycerides may have resulted from increased metabolism of alanine to pyruvate, and its incorporation into lipids under the stimulus of elevated insulin levels.

PIP: 23 women using various combined oral contraceptive (OC) preparations were studied for the effect of oral L-alanine loads on serum triglycerides and plasma insulin and glucose levels. Fasting serum triglyceride levels were significantly (p less than .001) higher in women taking OCs than in the 13 control subjects. OC users also had significantly (p less than .001) higher serum triglyceride levels at all test times following alanine loading than controls. There were no marked differences between the preparations used. Increases in plasma insulin after alanine loading were similar for users and nonusers, while glucose concentrations were unchanged. It is suggested that the alteration in serum triglycerides may have been due to an increase metabolism of alanine to pyruvate, and its incorporation into lipids under insulin stimulus.

Age-related changes in respiration coupled to phosphorylation. II. Cardiac mitochondria.

Age-related changes in mitochondrial adenine nucleotide metabolism may underlie the progressive decline in cardiac function. Oxidase activity coupled with phosphorylation, adenine nucleotide translocase (AdNT) activity, adenine nucleotide pool size and membrane lipid composition were determined using cardiac mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. While an age-associated 15% decrease in respiratory activity was not significant, AdNT activity of the aged rat was 20% lower (P less than 0.05) than that of the young rat. The exchangeable matrix adenine nucleotide pool (ATP + ADP) tended to decrease with age. In comparison to the young, membrane lipids of cardiac mitochondria from aged rat had a 43% higher (P less than 0.01) cholesterol/phospholipid-Pi ratio and a significantly lower (P less than 0.01) phosphatidyl ethanolamine/phosphatidyl choline ratio. The overall change in the fatty acid pattern of mitochondrial membrane lipids resulted in a significant (P less than 0.01) decrease in the n-6/n-3 fatty acid ratio. All values obtained for the mature rat fell between those of the young and aged rats. These data suggest that the reduced cardiac AdNT activity in the aged rat is a consequence of both a diminished pool of exchangeable adenine nucleotides and a lower AdNT velocity. Age-related changes in the lipid components of the membrane matrix in which the AdNT is embedded may underlie the decrease in respiratory activity.