Large Intracellular Pool Research Articles

A large intracellular pool of inactive Na channel alpha subunits in developing rat brain.

An intracellular pool of Na channel alpha subunits has been detected in developing brain cells in vivo and in vitro by phosphorylation with cAMP-dependent protein kinase, immunoprecipitation with specific antiserum, and NaDodSO4 gel electrophoresis or by radioimmunoassay. These alpha subunits are membrane-bound, contain complex carbohydrate chains, and have an apparent molecular weight of 260,000 like mature alpha subunits. In contrast to mature alpha subunits, the intracellular subunits are not covalently attached to a beta 2 subunit, and they do not bind saxitoxin with high affinity. They comprise 67-77% of the total immunoreactive alpha subunit in developing rat brain cells but are not a prominent component in the adult brain. It is proposed that this intracellular pool of alpha subunits forms a ready reserve of preformed subunits for incorporation into the surface membrane during periods of active membrane biogenesis. The results suggest that disulfide linkage of the alpha and beta 2 subunits, insertion into the cell surface membrane, and attainment of a functional conformation are closely related late events in the biogenesis of the Na channel. These processes may regulate the number of functional Na channels in the developing brain.

Proposed mechanism for increased insulin-mediated glucose transport in adipose cells from young, obese Zucker rats. Large intracellular pool of glucose transporters.

The mechanism for hyperresponsive insulin-mediated glucose transport in adipose cells from 30-day-old obese Zucker rats was examined. Glucose transport was assayed by measuring 3-O-methylglucose transport, and the concentration of glucose transporters was estimated by measuring specific D-glucose-inhibitable cytochalasin B binding. Insulin increased glucose transport activity by approximately 17 fmol/cell/min in cells from obese rats compared to 3 fmol/cell/min in lean littermates. Insulin increased the concentration of glucose transporters in the plasma membrane fraction by about 15 pmol/mg of membrane protein in both groups. The insulin-mediated decrease in the concentration of transporters in the low-density microsomal fraction was 30 pmol/mg of membrane protein for the obese rats compared to 15 pmol/mg of membrane protein for the lean controls. An estimated number of glucose transporters was calculated using membrane protein and enzyme recoveries for each group. Insulin increased the number of transporters in the plasma membrane by 3 X 10(6) sites/cell for the obese rats and only 0.6 X 10(6) sites/cell for the lean controls. In addition, insulin decreased the number of transporters/cell in the intracellular membrane pool by approximately 4 X 10(6) sites/cell for the obese rats and 0.9 X 10(6) sites/cells for the lean rats. The total number of transporters/cell was about 7 X 10(6) sites/cell for the obese animals and 1.6 X 10(6) sites/cell for the lean controls. In the basal state, more than 80% of these transporters were located in the intracellular pool for both the lean and obese rats. Thus, the marked hyperresponsive insulin-mediated glucose transport observed in adipose cells from 30-day-old obese Zucker rats may be the consequence of a marked increase in the number of glucose transporters in the intracellular pool.

Phospholipids as acyl donors to membrane proteins of Mycoplasma capricolum.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph, contains a large number of membrane proteins covalently modified by both saturated and unsaturated fatty acids (Dahl, C.E., Dahl, J.S., and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818). Pulse-chase experiments show that the radioactivity in the fatty acid moieties of the acyl proteins increases rather than decreases during a 4.5-h incubation period suggesting that a large intracellular pool of metabolites such as phospholipid serves as the donor for protein acylation. We find that cells incubated for 4 h in a growth medium containing [3H]palmitate-labeled phosphatidylglycerol or 2-[3H]palmitoyl dipalmitoylphosphatidylcholine show the same labeling pattern as cells incubated for 4 h in a complete growth medium with [3H]palmitate. Exogenously added phospholipids are not hydrolyzed to free fatty acid during the labeling period. Acylation of proteins is inhibited in cells treated with chloramphenicol showing that there is no pool of proacyl protein in the cell. Labeling of membrane proteins also occurs with [3H]glycerol. Glycerol is incorporated primarily into the same proteins as oleate suggesting that acylation by unsaturated fatty acid may involve a protein bound diglyceride moiety. Palmitate, on the other hand, appears to bind to other sites along the polypeptide chain in addition to the diglyceride moiety.

Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell. Apparent redistribution of receptors cycling between a large intracellular pool and the plasma membrane.

Previous studies have proposed that insulin increases the binding of insulin-like growth factor II (IGF-II) in isolated rat adipose cells at 24 degrees C by increasing receptor affinity (Ka). This study re-examines these observations under conditions in which receptor-ligand internalization is blocked by 1 mM KCN. In the absence of KCN, adipose cells bind 0.71 amol of IGF-II/cell with low apparent affinity (0.030 nM-1), of which greater than 75% is not accessible to trypsin. In contrast, in the presence of KCN, IGF-II binding is decreased by 95% and its apparent affinity increased to 0.21 nM-1. Moreover, greater than 60% of the bound IGF-II now is sensitive to trypsin. In either the absence or presence of KCN, approximately 20% of the cell's total IGF-II receptors are present in the plasma membranes and approximately 80% in the low density microsomes. Insulin induces a 5-fold increase in cell surface IGF-II receptors without a change in affinity when IGF-II binding is measured in the presence of KCN. Similarly, insulin increases IGF-II receptor concentration in the plasma membranes and concomitantly decreases that in the low density microsomes. Receptor affinity in these two subcellular membrane fractions is not affected by incubation of intact cells with either insulin or KCN and is similar to that observed in intact cells in the presence of KCN. Addition of KCN prior to insulin abolishes all of these effects of insulin. These data suggest that (a) the effects of KCN reflect a selective blockade of endocytosis; (b) in the absence of KCN, IGF-II binds to receptors of constant affinity that cycle between the plasma membrane and an intracellular pool resulting in an accumulation of intracellular IGF-II; (c) insulin induces an increase in IGF-II binding by causing a steady state redistribution of receptors from this intracellular pool to the plasma membrane; and (d) this redistribution in the intact cell can only be detected using Scatchard analysis when recycling of the receptors is prevented by KCN.

Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation.

Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.

Intracellular assembly of human fibrinogen.

Hep-G2 cells, pulse-labeled with L-[35S]methionine, incorporate radioactivity within 2 min into precursor forms of fibrinogen and into fibrinogen. Pulse-labeled intracellular fibrinogen is first composed of radioactive B beta chains, followed by nascent A alpha chains. Radioactive gamma chains accumulate in the cells and later contribute, via intermediate forms, to the assembly of fibrinogen. Following a pulse-chase incubation with L-[35S]methionine, the radioactive composition of newly secreted fibrinogen also reflects the fact that there is a large intracellular pool of gamma chains.

Human interferon-gamma is internalized and degraded by cultured fibroblasts.

Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes.

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes.

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.

Intracellular Pools of Thymidine Reduce the Antiviral Action of Acyclovir

Acyclovir inhibits herpes simplex virus (HSV) infections better in human fibroblasts than in green monkey kidney cells. Thymidine and acyclovir compete for the active site of HSV thymidine kinase. Large intracellular pools of thymidine found by high-performance liquid chromatography (HPLC) seemed to be responsible for the reduced activity of acyclovir. Such intracellular thymidine pools could not be measured previously, and the HPLC technique may help in evaluating the expected efficiency of acyclovir in cells and tissues prior to treatment.

102] Glycosylation of rhodopsin

This chapter discusses the glycosylation of rhodopsin. The oligosaccharide of rhodopsin is relatively simple, consisting of two separate chains, each composed of mannosc and N -acetylglucosa-mine. Structures of this type are synthesized on microsomal membranes as lipid-linked oligosaccharides which are transferred en bloc to an asparagine residue of the newly synthesized polypeptide. The transfer of galactose and fucose from the respective sugar nucleotides to rhodopsin can be demonstrated with bovine rod outer segment preparations. The site of this secondary glycosylation of opsin can be demonstrated by the use of another inhibitor in vitro . A large intracellular pool of uridine diphosphate (UDP)-N-acetylglucosamine is inferred from the relatively slow autoradio-graphic labeling of the rough endoplasmic reticulum. The synthesis of lipid-linked oligosaccharide intermediates has been described in chick embryo retina. Comparable oligosaccharide-lipids are formed by cell-free bovine retina preparations with evidence that they are also transferred to rhodopsin.

The effects of cycloheximide on the biosynthesis and secretion of proteoglycans by chondrocytes in culture.

Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulphate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t 1/2 less than 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t 1/2 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM-cycloheximide, but the incorporation of [35S]sulphate into proteoglycans was only inhibited gradually(t 1/2 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulphated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulphate and chased for up to 6 h in the presence of 1 mM-cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t 1/2 120 min to t 1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion, as well as inhibiting further protein core synthesis. The results suggested that the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulphate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labelled with [35S]sulphate showed an increase with time of both the average proteoglycan size and the length of the constituent chondroitin sulphate chain. However, the proportion of synthesized proteoglycans able to form stable aggregates did not alter.

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37°C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37°C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37°C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25°C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.

Phosphocreatine in Ehrlich ascites tumor cells detected by noninvasive 31P NMR spectroscopy

31P NMR spectra of intact Ehrlich ascites tumor cells of high phosphorylation potential reveal a well-defined resonance peak assignable to phosphocreatine, corresponding to 2.3 μmoles/ml cell H 2O in adenosine-treated cells containing 5.2 μmoles ATP/ml. The NMR spectrum of Ehrlich cells incubated with iodoacetate and glucose indicates depletion of phosphocreatine and ATP to undetectable levels and substantial accumulation of fructose-1,6-bisphosphate. From the difference between the chemical shifts of internal P i and phosphocreatine resonances, the intracellular pH was estimated to be 7.1 ± 0.1 in protein-synthesizing cells suspended in a medium of pH 7.4 at 10°C. Ehrlich cells are unable to transfer the labeled amidine group from L-(guanidino- 14C)-arginine to the large intracellular glycine pool to form labeled guanidinoacetate, the demethylated precursor of creatine. These results imply that the synthesis of phosphocreatine in ATP-rich Ehrlich cells is limited primarily by the extracellular free creatine supply, the extent of which depends upon the degree of cachectic perturbation of energy and nitrogen metabolism of the tumor-bearing host.

Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells.

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.

Dual pathway of B lymphocyte differentiation in vitro.

Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and theta surface markers and autoradiography after [3H] thymidine uptake. The changes observed were related to biochemical parameters such as incorporation of [3H] thymidine into DNA, Ig biosynthesis and secretion. Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig. Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with [3H] thymidine. We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.

Perturbation of Generation Cycle of Human Leukemic Myeloblasts In Vivo by Methotrexate

Abstract The cytokinetic effects of Methotrexate (MTX) on leukemic myeloblasts in vivo in man have been studied, utilizing in vitro and in vivo labeling with 3H-TDR, mitotic index, stathmokinetic index after VCR, measurements of single-cell DNA content, and in vitro labeling with 3H-UDR. It is demonstrated that MTX arrests cells in S for a period (about 20 hr) that corresponds to their DNA synthesis time. This S-phase arresting effect of MTX seems restricted to those cells that are in S at the time of MTX exposure. Cells in G1, G2, and M are not directly influenced by MTX. Some implications of these observations for combination drug therapy and for exploiting differences in the duration of cell cycle phases between leukemic blast cells and differentiating hemopoietic cells to obtain preferential killing of leukemic cells are briefly discussed. In one case, erythroblasts hardly incorporated exogenous TDR and UDR, although their mitotic activity was high. After MTX there was a transient but very pronounced increase in TDR and UDR ultilization. It is suggested that these cells may have had a large intracellular pool of thymidine triphosphate that was temporarily reduced by the action of MTX. Whether these erythroblasts were of leukemic origin remains undecided.

Orthophosphate turnover in the extracellular and intracellular space of mouse liver.

The flux rates of Pi from blood plasma into the extracellular and intracellular space of mouse liver have been measured in vivo with the [32P]Pi-tracer technique. The specific radioactivities of plasma Pi and total liver Pi have been measured 0.1–4 min after injection of carrier-free [32P]Pi. The results have been treated with the analogue computer technique. The main conclusions are: 1 The flux rate of Pi from blood plasma into the extracellular liver space amounts to 1.14 μmoles and that from the extracellular into the intracellular liver space to 0.39 μmoles per g fresh liver per min. 2 The influx of Pi through the liver cell membrane takes place without transient transformation into another phosphate compound rapidly exchangeable with large intracellular phosphate pools. 3 The specific radioactivity of intracellular Pi is somewhat higher than that of [γ-32P]ATP, thus indicating the absence of intracellular Pi-compartmentation with isotope kinetic relevance.

Pathogenesis of maple-syrup-urine disease: observations during dietary management and treatment of coma by peritoneal dialysis

Experiences with the management of a case of maple-syrup-urine disease from early infancy to death at 18 months of age are summarized. The use of a mixture of l-amino acids as the basis of a diet restricted in the branched-chain amino acids allowed the inclusion of a maximal amount of natural protein as the source of the essential branched-chain amino acids. The apparent importance of this natural source of the branched-chain amino acids for normal growth is emphasized. Myelination of the brain was normal on histological examination. The use of peritoneal dialysis for the treatment of coma which had not responded to vigorous intravenous therapy is discussed. Clear-cut neurological improvement occurred during dialysis without concomitant improvement in the metabolic acidosis. The branched-chain amino acids and their oxo acids did not account for the large negative base excess. Of plasma amino acids, mainly the branched-chain amino acids appeared in the dialyzate, with only modest decreases in the plasma levels. Thus, the plasma pool of branched-chain amino acids was being continously replenished by a large intracellular pool. A similar episode of coma occurring at 18 months of age was not so treated, and ended in death. The dramatic clinical and electroencephalographic response during dialysis and the apparently complete recovery which followed suggests that a diffusible “toxic” factor may be responsible for the coma of maple-syrup-urine disease, but does not itself establish that the branched-chain amino acids or their oxo acids are the etiological agents. The “toxic” factor may have little if anything to do with permanent structural changes and may result in coma and seizures in a brain that is apparently normal histologically. Further evaluation of these therapcutic approaches is warranted, but these exiguous results do not permit unbridled optimism.

Isosmotic intracellular regulation as a mechanism of volume control in crab muscle fibers

1. 1. Muscle fibers swollen in hypotonic sodium salines undergo spontaneous decreases in volumes (volume-readjustment) towards he initial volumes of the cells found in isotonic salines. 2. 2. This volume-readjustment is due to the elimination of osmotically active particles from the intracellular space of the muscle fibre. 3. 3. The only major intracellular solutes which were significantly lost from blue crab muscles as a result of volume-readjustment were the non-protein ninhydrin-positive substances. 4. 4. It is proposed that this component represents a fraction of the large intracellular pool of free amino acids found in crab muscle, and that free amino acids are being actively transported out of the muscles during volume-readjustment. 5. 5. Results of preliminary studies on the efflux of 2-aminoisobutyric acid-1- 14C from crab muscles are consistent with the view that amino acids are being actively transported out of the muscle cells during volume readjustment.

The pattern of disordered haemoglobin synthesis in homozygous and heterozygous beta-thalassaemia.

Summary:The rate of globin chain production has been studied in patients with homozygous β‐thalassaemia, heterozygous β‐thalassaemia, haemoglobin E‐thalassaemia, and sickle‐cell‐thalassaemia, and compared with that in non‐thalassaemic individuals. A partial or total deficit of β‐chain synthesis has been demonstrated in all forms of β‐thalassaemia. This results in the production of a large intracellular pool of α‐chains, the kinetics of which have been worked out. The α‐chains in this pool appear to contain haem and are unstable, rapidly becoming associated with the stromal fraction. These findings are examined in terms of the pathogenesis of the anaemia of thalassaemia.