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1.
Arch Biochem Biophys ; 436(1): 32-9, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15752706

ABSTRACT

Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arachidonic Acid/metabolism , Ascorbic Acid/metabolism , Cell Differentiation/physiology , Cyclic AMP/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Cell Division , Cells, Cultured , Cyclooxygenase Inhibitors/pharmacology , HL-60 Cells , Humans , Oxidation-Reduction , Vitamin D/metabolism
2.
Protoplasma ; 221(1-2): 11-7, 2003 May.
Article in English | MEDLINE | ID: mdl-12768337

ABSTRACT

Wistar rats were fed with different diets with or without supplement coenzyme Q(10) (CoQ(10)) and with oil of different sources (sunflower or virgin olive oil) for six or twelve months. Ubiquinone contents (CoQ(9) and CoQ(10)) were quantified in homogenates of livers and brains from rats fed with the four diets. In the brain, younger rats showed a 3-fold higher amount of ubiquinone than older ones for all diets. In the liver, however, CoQ(10) supplementation increased the amount of CoQ(9) and CoQ(10) in both total homogenates and plasma membranes. Rats fed with sunflower oil as fat source showed higher amounts of ubiquinone content than those fed with olive oil, in total liver homogenates, but the total ubiquinone content in plasma membranes was similar with both fat sources. Older rats showed a higher amount of ubiquinone after diets supplemented with CoQ(10). Two ubiquinone-dependent antioxidant enzyme activities were measured. NADH-ferricyanide reductase activity in hepatocyte plasma membranes was unaltered by ubiquinone accumulation, but this activity increased slightly with age. Both cytosolic and membrane-bound dicumarol-sensitive NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase, EC 1.6.99.2) activities were decreased by diets supplemented with CoQ(10). Animals fed with olive oil presented lower DT-diaphorase activity than those fed with sunflower oil, suggesting that the CoQ(10) antioxidant protection is strengthened by olive oil as fat source.


Subject(s)
Antioxidants/metabolism , Brain/metabolism , Fatty Acids/pharmacology , Liver/metabolism , Ubiquinone/pharmacology , Animals , Cell Membrane/enzymology , Cytosol/enzymology , Dietary Fats/pharmacology , Hepatocytes/metabolism , Liver/cytology , Male , NAD(P)H Dehydrogenase (Quinone)/metabolism , Olive Oil , Plant Oils , Rats , Rats, Wistar , Sunflower Oil , Ubiquinone/metabolism
3.
Protoplasma ; 217(1-3): 129-36, 2001.
Article in English | MEDLINE | ID: mdl-11732330

ABSTRACT

Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1 alpha,25(OH)2D3, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (delta psi m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1 alpha,25(OH)2D3-induced cells. However, ascorbate increased the mRNA levels of c-jun, junB, and c-fos in 1 alpha,25(OH)2D3-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1 alpha, 25(OH)2D3 is related to its effect on the cellular redox state and the modulation of AP-1 activity.


Subject(s)
Ascorbic Acid/pharmacology , Calcitriol/pharmacology , Cell Differentiation/drug effects , Monocytes/cytology , Transcription Factor AP-1/metabolism , Ascorbic Acid/chemistry , Cell Differentiation/physiology , DNA/metabolism , Ethanol/pharmacology , HL-60 Cells , Humans , Membrane Potentials/physiology , Mitochondria/metabolism , Monocytes/metabolism , Oxidation-Reduction , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Reactive Oxygen Species/metabolism
4.
Biochem J ; 331 ( Pt 1): 21-7, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9512457

ABSTRACT

1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1,25-(OH)2D3-induced differentiation of HL-60 cells can be explained by redox regulation of the cAMP pathway.


Subject(s)
Ascorbic Acid/metabolism , Calcitriol/pharmacology , Cyclic AMP/metabolism , HL-60 Cells/cytology , Signal Transduction , Cell Differentiation/drug effects , HL-60 Cells/metabolism , Humans , Oxidation-Reduction , Signal Transduction/drug effects
5.
J Bioenerg Biomembr ; 29(3): 259-67, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9298711

ABSTRACT

Serum provides cultured cells with survival factors required to maintain growth. Its withdrawal induces the development of programmed cell death. HL-60 cells were sensitive to serum removal, and an increase of lipid peroxidation and apoptosis was observed. Long-term treatment with ethidium bromide induced the mitochondria-deficient rho(o)HL-60 cell line. These cells were surprisingly more resistant to serum removal, displaying fewer apoptotic cells and lower lipid peroxidation. HL-60 cells contained less ubiquinone at the plasma membrane than rho(o)HL-60 cells. Both cell types increased plasma membrane ubiquinone in response to serum removal, although this increase was much higher in rho(o) cells. Addition of ubiquinone to both cell cultures in the absence of serum improved cell survival with decreasing lipid peroxidation and apoptosis. Ceramide was accumulated after serum removal in HL-60 but not in rho(o)HL-60 cells, and exogenous ubiquinone reduced this accumulation. These results demonstrate a relationship between ubiquinone levels in the plasma membrane and the induction of serum withdrawal-induced apoptosis, and ceramide accumulation. Thus, ubiquinone, which is a central component of the plasma membrane electron transport system, can represent a first level of protection against oxidative damage caused by serum withdrawal.


Subject(s)
Apoptosis , Cell Membrane/physiology , Ceramides/biosynthesis , Culture Media, Serum-Free/pharmacology , Ubiquinone/physiology , Cytosol/metabolism , DNA Replication/drug effects , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/metabolism , Enzyme Inhibitors/pharmacology , Ethidium/pharmacology , HL-60 Cells , Humans , Lipid Peroxides/metabolism
6.
Calcif Tissue Int ; 59(4): 277-82, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8781052

ABSTRACT

1,25 Dihydroxyvitamin D3 (calcitriol) induces differentiation of HL-60 leukemia cells. We studied the in vitro effect of a physiological concentration of ascorbate as potentiator of 1,25 dihydroxyvitamin D3 [(OH)2D3] activity by determining different markers of differentiation: nitroblue tetrazolium reduction, nonspecific esterase activity, and the expression of CD11b and CD14 surface antigens. Nitroblue tetrazolium reduction and nonspecific esterase activity increased up to 50% in the presence of both 1,25 (OH)2D3 plus 0.2 mM ascorbate (ASC), compared with (OH)2D3 as a unique agent. ASC also increased the expression of specific surface antigens (CD11b and CD14) during differentiation induced by 1,25 (OH)2D3, the effect being more pronounced after 48 hours of treatment with 10(-8) M 1,25 (OH)2D3. Furthermore, 1,25 (OH)2D3 alone increased intracellular cAMP level during differentiation, and the addition of ASC increased its concentration from 60 to 100% above the level reached with 1,25 (OH)2D3 as unique agent. ASC did not enhance the antiproliferative effect of calcitriol, suggesting that it only affects the ability of 1,25 (OH)2D3 to promote differentiation of HL-60 cells.


Subject(s)
Ascorbic Acid/pharmacology , Calcitriol/pharmacology , Monocytes/cytology , Cell Differentiation/drug effects , Drug Synergism , HL-60 Cells
7.
J Bioenerg Biomembr ; 26(4): 393-8, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7844114

ABSTRACT

Ascorbate, an essential nutrient in humans, primates, and guinea pig, is involved in many cellular functions. Ascorbate also modulates cell growth and differentiation. Ascorbate can reduce or stimulate the growth of tumor cells, depending on the cell type. The inhibitory effect is not specific for the biological active isomer L-ascorbate, and isoascorbate and D-ascorbate are more effective in reducing cell growth than L-ascorbate. These results indicate that ascorbate has a cytotoxic effect by killing cells directly, rather a cytostatic one. However, only L-ascorbate is able to stimulate cell growth, but the mechanism of this stimulation is still unknown. L-Ascorbate stimulates the in vitro differentiation of several mesenchyme-derived cell types by altering the expression of multiple genes as the cell progresses through specific differentiation programs. Stimulation of collagen matrix at gene transcription, mRNA stabilization, hydroxylation, and secretion is a key role for L-ascorbate. L-Ascorbate also prevents cell transformation by stabilization of the differentiated state and cooperates with other agents to induce differentiation in a leukemia cell line.


Subject(s)
Ascorbic Acid/pharmacology , Cell Differentiation/drug effects , Cell Division/drug effects , Growth Inhibitors/pharmacology , Animals , Ascorbic Acid/therapeutic use , Calcitriol/pharmacology , Cartilage/cytology , Cartilage/drug effects , Guinea Pigs , Humans , Leukemia, Promyelocytic, Acute/pathology , Mesoderm/drug effects , Mice , Neoplasms/drug therapy , Neoplasms/pathology , Nutritional Requirements , Oxidative Stress , Species Specificity , Tumor Cells, Cultured
8.
Leuk Res ; 17(10): 863-72, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8412299

ABSTRACT

The effects of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate on morphometric and stereological parameters have been studied using the HL-60 cell line as a differentiation model for the monocytic pathway. Evaluation of the differentiation was carried out by quantification of endoplasmic reticulum, Golgi apparatus, mitochondria and cytoplasmic granules. Changes in both nuclear and cytoplasmic volumes during TPA-induced differentiation led to a decrease of the nucleus-cytoplasmic ratio after 3 days of treatment. Plasma membrane glycoprotein pattern was also determined. The major change in cell surface was the presence of high amounts of glycoproteins containing N-acetyl glucosamine residues that make wheatgerm agglutinin lectin a valuable marker of the monocytic differentiation pathway in HL-60 cells.


Subject(s)
Leukemia, Promyelocytic, Acute/pathology , Tetradecanoylphorbol Acetate/pharmacology , Cell Differentiation/drug effects , Histocytochemistry , Humans , Lectins , Leukemia, Promyelocytic, Acute/metabolism , Membrane Glycoproteins/metabolism , Microscopy, Electron , Monocytes/pathology , Organelles/drug effects , Organelles/ultrastructure , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/ultrastructure
9.
J Bioenerg Biomembr ; 25(4): 411-7, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8226723

ABSTRACT

Plasma membranes isolated from rat liver by two-phase partition exhibited dehydrogenase activities for ascorbate free radical (AFR) and ferricyanide reduction in a ratio of specific activities of 1:40. NADH-AFR reductase could not be solubilized by detergents from plasma membrane fractions. NADH-AFR reductase was inhibited in both clathrin-depleted membrane and membranes incubated with anti-clathrin antiserum. This activity was reconstituted in plasma membranes in proportion to the amount of clathrin-enriched supernatant added. NADH ferricyanide reductase was unaffected by both clathrin-depletion and antibody incubation and was fully solubilized by detergents. Also, wheat germ agglutinin only inhibited NADH-AFR reductase. The findings suggest that NADH-AFR reductase and NADH-ferricyanide reductase activities of plasma membrane represent different levels of the electron transport chain. The inability of the NADH-AFR reductase to survive detergent solubilization might indicate the involvement of more than one protein in the electron transport from NADH to the AFR but not to ferricyanide.


Subject(s)
Electron Transport , Membrane Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , Animals , Cholic Acids , Coated Pits, Cell-Membrane/enzymology , Electrophoresis, Polyacrylamide Gel , Free Radicals , Liver/enzymology , Liver/ultrastructure , Membrane Proteins/isolation & purification , NADH Dehydrogenase/metabolism , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/isolation & purification , Oxidation-Reduction , Rats , Rats, Wistar , Wheat Germ Agglutinins/pharmacology
10.
Biochem Biophys Res Commun ; 192(2): 439-45, 1993 Apr 30.
Article in English | MEDLINE | ID: mdl-8484755

ABSTRACT

Besides its effect in inhibiting proliferation and inducing differentiation of HL-60 cells to macrophage-like cells, TPA also produces a transient increase of transplasma membrane redox activity and pyridine nucleotide levels and a shift in the NAD+/NADH ratio. After 24 h of incubation NADH ferricyanide reductase activity of isolated plasma membranes was significantly higher than that of plasma membrane from non-differentiated cells. This correlated with the enhanced short-term oxidation of NADH in response to ferricyanide by HL-60 cells incubated with TPA for 24 h. Since differentiated cells with similar levels of NADH showed different redox activities, the redox chain itself seems to be modulated during differentiation induced by TPA.


Subject(s)
Cell Differentiation , Cell Membrane/metabolism , Cell Line , Cell Membrane/enzymology , Electron Transport , Ferricyanides/metabolism , NAD/metabolism , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Tetradecanoylphorbol Acetate
11.
Biochem Biophys Res Commun ; 192(2): 707-13, 1993 Apr 30.
Article in English | MEDLINE | ID: mdl-8484777

ABSTRACT

Plasma membranes purified by two-phase partition from rat liver showed an NADH-ascorbate free radical reductase activity of about 14 nmoles NADH oxidized/min/mg protein. This activity was inhibited by N-ethyl maleimide, iodoacetate and iodoacetamide, reagents that covalently block thiol groups. NADH-ascorbate free radical reductase was also inhibited by reduced glutathione and the inhibitions observed with blocking reagents and reduced compounds were additive. These results support the involvement of sulphydryl groups in NADH-AFR reductase and point out the idea that a balance between reduced sulfhydryls and oxidized disulfides is required for the optimal function of this activity, considered as part of the transplasma membrane electron transport system.


Subject(s)
Liver/enzymology , NADH, NADPH Oxidoreductases/metabolism , Sulfhydryl Compounds/metabolism , Animals , Cell Membrane/enzymology , Ethylmaleimide/pharmacology , Free Radicals , Glutathione/pharmacology , In Vitro Techniques , Iodoacetamide/pharmacology , Iodoacetates/pharmacology , Iodoacetic Acid , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Oxidation-Reduction , Rats
12.
Biochim Biophys Acta ; 1073(2): 380-5, 1991 Mar 04.
Article in English | MEDLINE | ID: mdl-2009284

ABSTRACT

Ascorbate was maintained in the media during a long-term culture by HL-60 cells. The chemical oxidation of ascorbate was reversed in vitro by living HL-60 cells and was related to the amount of cells added. The increase of NADH concentration by lactate addition to cells was accompanied by an increase of both ascorbate regeneration and ferricyanide reduction. Further, plasma membrane enriched fractions from HL-60 cells revealed enhancement of both ascorbate regeneration and ferricyanide reduction in the presence of NADH when previously treated with detergent. The blockage of cell surface carbohydrates by wheat germ agglutinin (WGA) and Concanavalina ensiformis (Con A) lectins significantly inhibited the regeneration of ascorbate caused by the cells. These results support the idea that ascorbate is externally regenerated by the NADH-ascorbate free radical reductase as a part of the transplasma membrane redox system.


Subject(s)
Ascorbic Acid/metabolism , Cell Membrane/metabolism , Ascorbic Acid/pharmacology , Cell Count , Cell Division/drug effects , Cell Line , Concanavalin A/pharmacology , Dehydroascorbic Acid/metabolism , Ferricyanides/metabolism , Free Radicals , Humans , Kinetics , Lactates/metabolism , Lactic Acid , NAD/metabolism , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Oxygen Consumption , Wheat Germ Agglutinins/pharmacology
13.
Cancer Res ; 50(18): 5887-91, 1990 Sep 15.
Article in English | MEDLINE | ID: mdl-2393860

ABSTRACT

Ascorbate free radical stimulates the growth of human promyelocytic leukemia cells (HL-60) in the presence of a limited amount of serum (1%) when added to the cells under conditions where it is impermeable. Maximum growth stimulation occurs at concentrations from 5 x 10(-9) to 2 x 10(-8) M. Ascorbate mimicks the stimulation effect of its free radical but stimulates at higher concentrations. Autoxidation of ascorbate by oxygen produces its free radical, which apparently causes growth stimulation. Ascorbate could be regenerated by intact cells in vitro, since prevention of autoxidation of ascorbate in the presence of cells is observed. Neither dehydroascorbate nor isoascorbate increases HL-60 cell growth. Short term incubation of cells in the presence of ascorbate free radical induced intracellular NADH oxidation. We propose that the stimulation of growth of HL-60 cells shown here could be caused by activation of the transplasma membrane electron transport system by the ascorbate free radical.


Subject(s)
Ascorbic Acid/pharmacology , Leukemia, Promyelocytic, Acute/pathology , Ascorbic Acid/metabolism , Free Radicals , Humans , Leukemia, Promyelocytic, Acute/metabolism , NAD/metabolism , Oxidation-Reduction , Tumor Cells, Cultured
14.
Biochem Biophys Res Commun ; 154(3): 1029-33, 1988 Aug 15.
Article in English | MEDLINE | ID: mdl-3408483

ABSTRACT

Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free radical reduction by NADH of 4-5 nmoles of oxidized NADH/min/mg protein. This activity was inhibited 80% by ConA and up to 97% by WGA and LFA lectins. NADH-ascorbate free radical reductase was also inhibited in rat liver plasma membranes preincubated with neuraminidase or trypsin, but no additional inhibition was observed in the presence of LFA after enzyme digestion. It appears that the integrity of glucan moieities of the cell surface glycoconjugates are necessary for the optimal function of this activity that could be considered as part of the transplasma membrane electron transport system.


Subject(s)
Cell Membrane/physiology , Glycoconjugates/physiology , NADH, NADPH Oxidoreductases/metabolism , Animals , Cell Membrane/enzymology , Kinetics , Lectins/pharmacology , Liver/enzymology , Neuraminidase/pharmacology , Rats
16.
Article in English | MEDLINE | ID: mdl-2907432

ABSTRACT

1. Sodium pump, measured as K+-dependent, ouabain sensitive pNPPase, but not the non-specific, ouabain insensitive pNPPase, was stimulated by thyroxin in epidermis of tadpoles of Rana perezi. 2. Epidermal K+-pNPPase of thyroxin treated tadpoles was only stimulated in those stages already showing activity and reached levels similar to adult frogs in tadpoles at metamorphic climax.


Subject(s)
Epidermis/metabolism , Metamorphosis, Biological , Ranidae/physiology , Sodium/metabolism , Thyroxine/physiology , Animals , Epidermis/physiology , Larva , Phosphoric Monoester Hydrolases/analysis , Ranidae/metabolism
17.
Eur J Cell Biol ; 44(2): 176-86, 1987 Oct.
Article in English | MEDLINE | ID: mdl-2826167

ABSTRACT

Aqueous two-phase partition and preparative free-flow electrophoresis were used in series to isolate the plasma membranes of amphibian epidermis. Fractions obtained by two-phase partition were 40-fold enriched in a K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase relative to the total homogenate and based on morphology were representative isolates of all epidermal cells together. Small mucosal granules and mucin aggregates were the primary contaminants. Based on activities of marker enzymes, contents of mitochondria, Golgi apparatus and endoplasmic reticulum were low (0.15 that of total homogenate) or absent. When plasma membranes isolated by aqueous two-phase partition were subjected to preparative free-flow electrophoresis, they were distributed toward the anode in a series of fractions of increasing net negative charge, sialic acid content and specific activity of the K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase reminiscent of the activity gradient from base to apex for frog epidermis observed from cytochemical investigations. The most electronegative fractions nearest the anode and to the left of the main protein peak were enriched in both sulfate groups and thick membranes of the stratum corneum. A fraction migrating less toward the anode and to the right of the main protein peak contained hemidesmosomes together with the lowest enrichments of sialic acid, sulfate and the phosphatase. The results suggest that the plasma membranes isolated from mixed cell populations, such as those encountered in epidermal homogenates, may be resolved by free-flow electrophoresis according to cell type of origin following activity gradients present in the original tissue. Additionally, the findings provide independent biochemical confirmation of a base-to-apex gradient of transport (ATPase) activity associated with the plasma membranes of cells of the different strata of the amphibian epidermis.


Subject(s)
Cell Membrane/physiology , Epidermis/ultrastructure , 4-Nitrophenylphosphatase/metabolism , Animals , Cell Fractionation , Cell Membrane/analysis , Electrophoresis , Endoplasmic Reticulum/analysis , Epidermis/analysis , Golgi Apparatus/analysis , Histocytochemistry , Microscopy, Electron , Mitochondria/analysis , Potassium/pharmacology , Rana pipiens , Sialic Acids/analysis , Sulfates/analysis
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