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1.
Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin. 相似文献
2.
Robert S. Slaughter Raymond G. Fenwick Eugene M. Barnes 《Archives of biochemistry and biophysics》1981,211(1):494-499
The transport of thymidine and hypoxanthine was investigated in mutant Chinese hamster lung fibroblasts deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Kinetic data from rapid uptake experiments (0.5–4.5 s) indicate that thymidine is transported by a monophasic saturable system (Km = 0.29 mM, V = 6.7 nmol/min · mg) which is competitively inhibited by hypoxanthine (Ki = 3.3 mM). The cells displayed a single transport system for hypoxanthine (Km = 2.0 mM, V = 8.9 nmol/min · mg) that is inhibited competitively by thymidine (Ki = 0.43 mM). Both hypoxanthine and thymidine entry were noncompetively inhibited by nitrobenzylthioinosine, but thymidine transport was more sensitive. A kinetic model in which hypoxanthine and thymidine share a common transporter can account for the competitive inhibition and the observation that the inhibition constants are similar to the Michaelis constants. 相似文献
3.
In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4. 相似文献
4.
The regulation of hypoxanthine transport activity by Chinese hamster lung fibroblasts grown in culture was examined in wild-type clones and 8-azaguanine-resistant mutant clones which lack hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine transport activity increases with increased rates of cellular growth expressed as viable cell number, total cell protein, and DNA synthesis. The transport activity for hypoxanthine declines when the fibroblasts approach confluence or after exposure to cycloheximide or actinomycin D. In vivo incubation of either fibroblast subline with 100 μm dibutyryl cyclic AMP decreases transport activity over 50%, whereas exposure to 10 μm dibutyryl cyclic GMP increases hypoxanthine uptake by 40%. A synergistic effect is observed when fibroblasts are incubated with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine or theophylline) plus glucagon, an adenylate cyclase stimulator. Such additions result in a 70% decrease in the cellular transport capacity. Stimulation of hypoxanthine transport by 40% is observed following incubation with insulin. Addition of all agents produces maximum changes in the rate of hypoxanthine transport only after a 6-h in vivo incubation with the fibroblasts. These findings suggest that hypoxanthine transport is regulated by the intracellular concentration of cyclic nucleotides. This control may occur at the level of gene expression for a hypoxanthine transport protein. 相似文献
5.
It has been determined that cortisol and a few other steroids are transported outward from certain mammalian cells growing in vitro. The extrusion process is temperature dependent, glucose dependent, saturable, and operates for only a few selected steroids. Many, but not all, steroids are able to block the extrusion process but are not themselves transported. The outward transport process for steroids has been found in mouse fibroblasts, mouse lymphoma cells, and functional mouse adrenal gland tumor cells growing in vitro. The transport process is not present in two varieties of cells cultured from human sources—HeLa or diploid fibroblasts, WI-38. 相似文献
6.
Adenine, guanine, and hypoxanthine were rapidly incorporated into the acid-soluble nucleotide pool and nucleic acids by wild type Novikoff cells. Incorporation followed normal Michaelis-Menten kinetics, but the following evidence indicates that specific transport processes precede the phosphoribosyltransferase reactions and are the rate-limiting step in purine incorporation by whole cells. Cells of an azaguanine-resistant subline of Novikoff cells which lacked hypoxanthine-guanine phosphoribosyltransferase activity and failed to incorporate guanine or hypoxanthine into the nucleotide pool, exhibited uptake of guanine and hypoxanthine by a saturable process. Similarly, wild type cells which had been preincubated in a glucose-free basal medium containing KCN and iodoacetate transported guanine and hypoxanthine normally, although a conversion of these purines to nucleotides did not occur in these cells. The mutant and KCN-iodoacetate treated wild type cells also exhibited countertransport of guanine and hypoxanthine when preloaded with various purines, uracil, and pyrimidine nucleosides. The cells also possess a saturable transport system for uracil although they lack phosphoribosyltransferase activity for uracil. In the absence of phosphoribosylation, none of the substrates was accumulated against a concentration gradient. Thus transport is by facilitated diffusion (nonconcentrative transport). Furthermore, the apparent Km values for purine uptake by untreated wild type and azaguanine-resistant cells were higher and the apparent Vmax values were lower than those for the corresponding phosphoribosyltransferases... 相似文献
7.
Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. Here we report the functional characterization of this transporter in human fibroblasts. Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake. Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency. In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+. Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine. Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters. A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range. These result indicate the presence of multiple types of carnitine transporters in human cells. 相似文献
8.
The synteny of human mannose phosphate isomerase and pyruvate kinase and the assignment of the genes for these two enzymes to chromosome 15 were confirmed by analysis of 43 independently derived human-mouse hybrid clones. Hybrids between mouse cells deficient in hypoxanthine-guanine phosphoribosyltransferase and human fibroblasts carrying an X/15 chromosome translocation were also included in this study. 相似文献
9.
The preferential interaction of L-threonine with transport system ASC in cultured human fibroblasts.
O Bussolati B M Rotoli P C Laris V Dall'Asta G C Gazzola 《Biochimica et biophysica acta》1991,1070(2):305-312
The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts. 相似文献
10.
B M King T Rozaklis J J Hopwood G S Harper 《Biochemical medicine and metabolic biology》1992,47(3):260-264
The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts. 相似文献
11.
The characteristics of the uptake of L-cystine by LLC-PK1 cells were examined. The uptake diminished with time in culture after passage of cells while the uptake of sugar increased. In 48-h-cultured cells at a range of cystine concentrations including physiological levels uptake occurred via a saturable process which was independent of medium sodium concentration and pH. No inhibition of cystine uptake occurred in the presence of lysine which is known to share the cystine transport system in uncultured renal proximal tubule cells and brush-border membrane vesicles. Glutamate was a potent inhibitor of cystine uptake and participated in heteroexchange diffusion with cystine. The cystine-glutamate transport process resembles that of cultured human fibroblasts. The inability of these cells to reflect the genetically determined cystine-lysine system which is altered in the kidney in human cystinuria makes them an inappropriate model of the renal tubule cell cystine transport system. On the other hand, they may provide a model system for examining the factors which determine the presence of the various cystine transport process. 相似文献
12.
N S Magnuson L E Perryman D P Suttle J L Robinson P H Mason K M Marta 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,81(3):781-786
In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein). 相似文献
13.
S L Zhou D Stump D Sorrentino B J Potter P D Berk 《The Journal of biological chemistry》1992,267(20):14456-14461
A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids. 相似文献
14.
X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: Detection of heterozygotes by selective medium 总被引:8,自引:0,他引:8
Barbara Ruben Migeon 《Biochemical genetics》1970,4(3):377-383
Skin fibroblasts from five unrelated males with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency and from their families have been exposed to medium containing 6-thioguanine. This purine analogue selects against cells with normal hypoxanthine-guanine phosphoribosyl transferase activity and therefore permits detection of mutant cells in heterozygous populations. The results of these studies are compared to those obtained by autoradiography of single-cell clones of skin fibroblasts from the same subjects. In each case, the results of the selective method are similar to those obtained by clonal analysis. The use of selective medium therefore provides a sensitive means to detect heterozygosity at this locus and may provide a general method to select cells with X-linked markers from heterozygous populations.This work was supported by grants from the U.S.P.H.S. (#HD 00486), The Joseph P. Kennedy, Jr., Memorial Fluid Research Fund, and the National Foundation for Neuromuscular Diseases, Inc. 相似文献
15.
The uptake of hypoxanthine by Chinese hamster lung fibroblasts grown in tissue culture was studied in wild type clones and 8-azaguanine-resistant mutant clones devoid of hypoxanthine-guanine phosphoribosyltransferase. Wild type fibroblasts rapidly accumulate [3H]hypoxanthine from the medium and over 80% of the intracellular radioactivity is found in acid-soluble nucleotides. The phosphoribosyltransferase-deficient clones accumulate much lower levels of hypoxanthine and over 85% of the intracellular 3H label is associated with chemically unaltered hypoxanthine. The internal level of hypoxanthine in the mutant clones rapidly approaches but does not exceed that present in the medium. Wild type and phosphoribosyltransferase-deficient cells take up hypoxanthine at almost identical initial rates at external hypoxanthine levels from 2 to 300 muM. Analysis of these data reveals two transport systems that obey the Michaelis-Menten relationship. These differ markedly in affinity, yielding average Km values of 20 and 600 muM for both cell types. Hypoxanthine transport by both low and high affinity transport systems is blocked by p-chloromercuriphenylsulfonate and N-ethylmaleimide. Counter-transport of hypoxanthine was demonstrated in phosphoribosyltransferase-deficient fibroblasts. It is concluded that hypoxanthine is transported into Chinese hamster cells by means of carrier-mediated processes (facilitated diffusion) that operate independently of phosphoribosylation. 相似文献
16.
We have used [3H]guanine incorporation as a rapid and sensitive assay of xanthine-guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent. 相似文献
17.
C.H.M.M. de Bruyn T.L. Oei P. Hösli 《Biochemical and biophysical research communications》1976,68(2):483-488
An ultra-microchemical method using radioactive substrates has been developed for enzyme activity measurements at the single cell level. In order to demonstrate the possibilities of this radiochemical microassay, activity measurements of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) and of adenine phosphoribosyl transferase (A-PRT) in isolated human fibroblasts are described. There was a linear relationship between the number of cells incubated and the enzyme activities found. It was observed that the HG-PRT activity in single, skin derived, fibroblasts did not differ from that in amniotic fluid derived fibroblasts, thus providing a new, quantitative assay for rapid prenatal diagnosis in the Lesch-Nyhan syndrome. 相似文献
18.
We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of Km. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process. 相似文献
19.
P. Hösli C. H. M. M. de Bruyn F. J. J. M. Oerlemans M. Verjaal R. E. Nobrega 《Human genetics》1977,37(2):195-200
Summary A previously developed simple ultramicromethod has been used for the rapid prenatal diagnosis of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) deficiency. The method is based on the incubation of small numbers of visually selected, lyophilized fibroblasts (in the present study five cells per incubation) with radioactive substrate in an end volume of 0.3 l. Fibroblasts derived from the amniotic fluid of a 15-week male fetus in a heterozygote for the X-linked Lesch-Nyhan syndrome showed a severe degree of HG-PRT deficiency. In total 50 fibroblasts were used. The diagnosis was confirmed upon termination of the pregnancy by the demonstration of HG-PRT deficiency in fetal erythrocytes and cultured skin fibroblasts.HG-PRT: hypoxanthine-guanine phosphoribosyl transferase 相似文献
20.
Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling 总被引:86,自引:50,他引:36
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《The Journal of cell biology》1980,85(3):839-852
Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors. 相似文献