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C R Park

Publications and source records attributed to C R Park.

At least 37 records · Page 2Linked to original sources
PubMed1984

Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein.

This study extends our earlier work (Abumrad, N. A., Perkins, R.C., Park, J.H., and Park, C.R. J. Biol. Chem. 256, 9183-9191) which showed that oleate permeates the plasma membrane of the rat adipocyte principally by a transport process with the characteristics of facilitated diffusion. In the present study, fatty acid (FA) transport is characterized with regard to its specificity and susceptibility to inhibition by protein modifiers. The kinetics of competitive inhibition for transport of oleate and stearate are shown under conditions where complications due to competition for binding of FAs to the albumin in the medium are minimized. Stearate inhibits influx of tracer oleate with a Ki that closely approximates its Km and, conversely, oleate inhibits similarly the influx of tracer stearate. Specificity of the FA transport system is shown in studies using a variety of natural FAs of different chain length, or FA analogues. Oleate (Km = 0.06 microM), stearate (Km = 0.16 microM), linoleate (Km = 0.22 microM), palmitate, (Km = 0.2 microM), and laurate (Km = 1.5 microM) are good substrates, but octanoate is not transported. An oxazolidine ring on C-5 but not on C-16 of stearate blocks binding to the transporter. Methylation of the carboxyl function but not alpha-bromination inhibits transport. These studies suggest that a FA must have a hydrocarbon chain of at least nine carbons and a free carboxyl function to be recognized by the transporter. FA transport does not require Na or ATP. Pronase but not trypsin treatment of intact cells reduces fatty acid influx. Transport is insensitive to maleimides. It is strongly and irreversibly blocked by pretreatment of the cells with the stilbene compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, but only slightly inhibited by dipyridamole. Polyacrylamide gel electrophoresis of plasma membrane proteins from cells treated with [3H] 4,4'-diisothiocyanostilbene-2,2'-disulfonate shows a peak of radioactivity at about Mr = 85,000. When cells are incubated in various concentrations of this agent, the counts recovered in the peak reach a maximum coincident with maximum inhibition of transport. We conclude that permeation of the plasma membrane of the adipocyte by long-chain FAs at physiological concentrations is mediated by a protein transporter with distinct specificity requirements.

Adipose Tissue↗
PubMed1982

Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids.

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.

Binding, Competitive↗
PubMed1981

15N- and 2H-substituted maleimide spin labels: improved sensitivity and resolution for biological EPR studies.

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of (15)N for (14)N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [(2)H]MSL and the (15)N-substituted and deuterated derivative [(15)N, (2)H]MSL were compared with those of the parent MSL. The [(15)N, (2)H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (i) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (ii) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing (14)N with (15)N. In the freely tumbling state, the spectra of [(15)N, (2)H]MSL and [(2)H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD(+) oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [(15)N, (2)H]MSL and [(2)H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [(15)N, (2)H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 musec to 1 msec. The EPR spectrum of [(15)N, (2)H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and g tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the (15)N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by (15)N substitution. Use of (15)N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.

Computers↗
PubMed1980

Enhancement of free radical reduction by elevated concentrations of ascorbic acid in avian dystrophic muscle.

It has been postulated that the degenerative process in dystrophic muscle results from increased concentrations of free radicals, peroxides, or lipid hydroperoxides. Therefore, the reduction of the free radical tanol (2,2,6,6-tetramethyl-4-piperidinol-1-oxyl) by extracts of muscles of dystrophic and normal chickens was studied. Pectoral (white) and thigh (red) muscles were used. For initial rate measurements, the various muscle extracts were added to an equal volume of 0.2 mM tanol. Reaction mixtures were introduced into the EPR cavity in a standard aqueous flat cell. Rates were measured by continuously monitoring the decrease in signal amplitude of the center (MI = 0) solution tanol EPR resonance line (in-phase first harmonic absorption signal). With extracts from dystrophic white muscle, the reduction rate was 75% faster than normal, whereas in dystrophic red muscle extracts the rate was normal. This agreed with previous observations that white muscle is more severely affected than red in dystrophic chickens. The primary reductant was identified as reduced ascorbic acid, and the rate of reduction of tanol correlated directly with the concentrations of ascorbic acid in the various muscle extracts as shown by chemical analysis. The results suggest an involvement of the intracellular redox status in the pathogenesis of avian muscular dystrophy.

Animals↗
PubMed1979

Mechanism of action of penicillamine in the treatment of avian muscular dystrophy.

Penicillamine, a cysteine analog with a reduced sulfhydryl group, has been used in this laboratory for the treatment of hereditary avian dystrophy. The drug delays the onset of symptoms and alleviates the debilitating aspects of the disease. To study the mechanism of drug action, the effects of penicillamine on white and red muscles of dystrophic chickens were examined with regard to the specific activities of the soluble enzymes glyceraldehyde-3-phosphate dehydrogenase, acetylphosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, glutathione preoxidase, superoxide dismutase, and catalase. The sulfhydryl contents of the soluble proteins and the concentration of myoglobin were also determined. In white dystrophic muscle (pectoral), there were large alterations in the various enzymatic activities compared to normal levels. In the DISCUSSION, these changes are related to the pathogenesis of the disease and to the adaptive response for protection of the severely affected fast fibers. Red dystrophic muscles (thigh) were minimally involved, in accordance with the known sparing action of the slow fiber type. The results suggested that the disease process in dystrophic muscle may be due to oxidation of the essential sulfhydryl groups of proteins. Penicillamine may produce therapeutic effects by altering the intracellular redox status, thereby promoting better regulation of enzymatic activity, membrane stability, and improved muscle function.

Animals↗
PubMed1978

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.

Adenosine↗
PubMed1978

Control of hepatic glucose output by glucagon and insulin in the intact dog.

The regulation of hepatic glucose production by glucagon and insulin has been studied in the intact dog. An attempt has been made to evaluate the role of basal physiological concentrations of the hormones in the regulation of glycogenolysis and gluconeogenesis. Somatostatin was infused continuously into postabsorptive dogs to inhibit the secretion of both glucagon and insulin. Either or both hormones were then replaced intraportally by continuous infusion as desired. The main observations were as follows. (1) When both hormones were simultaneously replaced for periods up to 4.5h, plasma insulin and glucagon concentrations, total glucose output (glycogenolysis plus gluconeogenesis), glucose utilization and the plasma glucose concentration closely matched the same parameters in 0.9% NaCl-infused controls. (2) When glucagon alone was infused, thereby creating a selective insulin deficiency, glucose output (primarily glycogenolysis) rapidly increased by as much as threefold. Glycogenolytic glucose production then fell off progressively and returned to the control value within 4h. The gluconeogenic conversion of [14C]alanine and [14C]lactate into [14C]glucose was stimulated markedly and increased progressively throughout the test period. Glucagon therefore converted the liver from an organ largely dependent on glycogenolysis for glucose production to one heavily dependent on gluconeogenesis. The potent inhibitory effect of basal insulin on postabsorptive glucose output was also clearly apparent. (3) When insulin alone was infused, thereby creating a selective glucagon deficiency, glucose output (glycogenolysis) fell abruptly by about 30% and remained decreased. Gluconeogenesis also decreased (20%) after the selective removal of both insulin and glucagon, but it only remained suppressed for 1h. The low glucose output led to a modest fall in the blood glucose concentration. Thus glucagon plays an important role in maintaining basal glucose production. (4) When insulin was infused and the plasma glucose was kept at its control concentration by infusion of glucose in similar experiments to the above, the hepatic output of glucose fell by as much as 75%. This demonstrates the presence of a glucagon-independent metabolic reflex triggered by a low plasma glucose concentration, the purpose of which is to maintain glucose output at a rate capable of preventing castastrophic hypoglycaemia.

Animals↗
PubMed1976

Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes.

The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.

Adrenergic alpha-Agonists↗
PubMed1976

Transport of D-allose by isolated fat-cells: an effect of adenosine triphosphate on insulin stimulated transport.

D-allose, a glucose analogue, is not metabolized by isolated fat-cells and its distribution space at equilibrium in the cells is the same as that of triated water. Uptake of allose is inhibited by glucose and 3-O-methylglucose, stimulated by insulin and virtually eliminated by cytochalasan B. Counter transport of allose out of fat-cells against a concentration gradient can be induced by exogenous glucose but not by pyruvate. It is concluded that allose is transported into fat-cells by the same carrier mediated transport system as glucose and that it is a suitable analogue with which to study the glucose transport system. Insulin stimulated allose transport, into or out of the cell, but not basal transport, is inhibited by a brief exposure of isolated fat-cells to exogenous ATP or ADP (but not AMP or AMP-PNP). The antilipolytic effect of insulin is not affected. The ATP inhibition is slowly reversible. It is suggested that ATP phosphorylates a membrane component and thereby blocks transmission of signal from the insulin receptor to the carrier system. Indirect evidence suggests that ATP does not alter the affinity of the insulin or glucose binding sites. Insulin decreases the Km of glucose metabolism of CO2 and lipid in isolated fat-cells and increases the Vmax. However,the hormone has no effect on the Ki of glucose as an inhibitor of allose transport. The glucose analogue, 3-O-methyl-glucose, also inhibits both glucose metabolism and allose transport. The Ki for both these processes is similar and is not affected by insulin. These results support the view that the effect of insulin on glucose transport is to raise the Vmax without a change in the Km. It appears further that sugar transport is not the major rate limiting step in metabolism at high glucose concentrations in the absence of insulin, or at most glucose concentrations in the presence of the hormone.

Adenosine Diphosphate↗
PubMed1976

Guanosine 3':5'-cyclic monophosphate binding proteins in rat tissues.

Rat tissues were surveyed for proteins which bind cGMP. Binding activity was high in extracts of lung, cerebellum, and small intestine, but was low in those of liver, adipose tissue, and skeletal muscle. DEAE-cellulose chromatography resolved two peaks of cGMP-binding activity in most tissues. The binding protein in peak 1 was eluted in the flow-through volume and was most abundant in extracts of intestine. It had a sedimentation coefficient of 6S and was highly specific for cGMP at pH 7.0 (dissociation constant KD=0.05 muM). No cGMP-dependent histone kinase activity was found for this peak. The binding protein in peak 2 was eluted by 0.05-0.15 M NaCl and was the predominant binding substance in lung, cerebellum, and heart. It had a sedimentation coefficient of 8S and binding was also highly specific for cGMP, with a KD of 0.05 muM. This peak of binding activity was associated with cGMP-dependent protein kinase activity which could be purified approximately 200-fold by Sepharose 6B chromatography. Cyclic GMP dependency of kinase activity was observed only at low histone concentrations. The abundance of one or both the above binding proteins correlated with the known basal levels of cGMP in the tissues.

Animals↗
PubMed1976

Carbohydrate metabolism in perfused livers of adrenalectomized and steroid-replaced rats.

In livers from fasted rats perfused with bicarbonate buffer containing bovine albumin and erythrocytes, adrenalectomy decreased glycogen levels and glucose production, impaired the incorporation of 14C from [14C]lactate into glucose or glycogen, and decreased the activity of the active (I) form of glycogen synthase. Cortisol treatment restored gluconeogenesis after 1 h and glycogen synthesis after 2 h. Adrenalectomy did not alter the production of glucose or lactate or the levels of gluconeogenic intermediates in livers from fasted rats perfused with fructose, but reduced the formation of glycogen from this substrate. Adrenalectomy increased the levels of lactate and decreased the levels of P-pyruvate and subsequent intermediates in the gluconeogenic pathway. These changes were reversed by cortisol treatment. It is concluded that glucocorticoids support gluconeogenesis and glycogen synthesis in livers from fasted rats primarily by facilitating a reaction(s) located between pyruvate and P-pyruvate in the gluconeogenic pathway and by promoting the conversion of inactive to active glycogen synthase.

Adenosine Diphosphate↗
PubMed1975

Stimulation by glucagon of the incorporation of U-14C-labeled substrates into glucose by isolated hepatocytes from fed rats.

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.

Acetates↗
PubMed1975

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.

Adenylyl Cyclases↗