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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ecto-Mg-ATPase isolated from chicken gizzard smooth muscle was solubilized, purified and characterized. The purification did not require the use of expensive or specialized apparatus. The chromatographic and electrophoretic characteristics of the ecto-Mg-ATPase from chicken are similar to those reported earlier for the ecto-Mg-ATPase isolated from rabbit skeletal muscle transverse tubule membranes [1992, J. Biol. Chem. 267, 11777-11782]. One obvious difference found was that the solubilized chicken ecto-Mg-ATPase can be stimulated approximately 1900% by the lectin Concanavalin A (Con A) under the same conditions that the rabbit enzyme is inhibited by approximately 50%. This stimulatory effect of Con A is useful for following the purification, and also increases the specific activity of the chicken enzyme to a very high level similar to that observed for the rabbit enzyme. After purification of the solubilized chicken ecto-Mg-ATPase by three steps of anion exchange chromatography, as well as Con A and erythroagglutinating Phaseolus vulgaris (PHA-E) lectin affinity chromatographies, a single diffuse glycoprotein band at approximately 66 kDa is observed after SDS-PAGE. This protein could be deglycosylated to a core protein of 53 kDa. Thus, the chicken gizzard protein is very similar in molecular size to the rabbit skeletal muscle ecto-Mg-ATPase both before and after deglycosylation [1992, J. Biol. Chem. 267, 11777-11782]. The N-terminal sequence of the 66 kDa chicken gizzard protein was found to be: Ala-Arg-Arg-Ala-Ala-Ala-Val-Leu-Leu-Leu-Leu-Ala. This is a unique sequence which, while very different from the rabbit ecto-Mg-ATPase N-terminus, exhibits some of the same characteristics, since it contains basic residues as the second and third amino acids, with the remainder of the N-terminus being very hydrophobic in nature. Furthermore, the chicken gizzard ecto-Mg-ATPase can be separated from the adhesion molecule, truncated cadherin (T-cadherin) by anion exchange chromatography, and is therefore not identical to that protein, as had been recently proposed [1993, Arch. Biochem. Biophys. 303, 32-43].
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PMID:Purification and characterization of the ecto-Mg-ATPase of chicken gizzard smooth muscle. 798 47

1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle15]-gastrin. 2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides. 3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55-61). The remaining 7 peptides were derived from the beta-subunit of the gastric H+/K(+)-ATPase. 4. The gastrin-binding activity remained in association with p78, and could be separated from the beta-subunit of the gastric H+/K(+)-ATPase, during chromatography on tomato lectin-Sepharose. 5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.
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PMID:Isolation and partial amino acid sequence of a 78 kDa porcine gastrin-binding protein. 801 37

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for measuring protein expression at the cell surface. 812 1

Insulin receptor number and insulin responsiveness were compared in a chicken hepatoma cell line (LMH) and in normal chicken hepatocyte (cHep) cells cultured in the same conditions. LMH cells expressed two- to threefold more insulin receptors than cHep cells, without significant changes in affinity. The tyrosine kinase activity of solubilized and lectin (lentil+wheat germ agglutinin; WGA)-purified LMH receptors was higher than that of cHep receptors. The ATP hydrolytic activity previously observed in WGA-purified receptors from chicken liver membranes was also present in WGA-purified receptors from cultured cHep cells. This unidentified membrane-associated ATPase was absent from LMH membrane-solubilized material and therefore from WGA-purified LMH insulin receptors. Finally, LMH cells incorporated at least tenfold more amino isobutyric acid than cHep cells in the absence of insulin and were more responsive to insulin. The enhanced basal amino acid transport of LMH cells was most probably the consequence of their proliferative activity. The enhanced insulin responsiveness of LMH cells can be accounted for, at least in part, by one or several of the modifications presently demonstrated in LMH cells when compared with normal cultured hepatocytes: increased insulin receptor number and tyrosine kinase activity and possibly the loss of the membrane-associated ATPase.
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PMID:Increased insulin receptor number and insulin responsiveness in a chicken hepatoma cell line. 813 46

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Clathrin-coated vesicles (CCV) were isolated from the rat renal cortex utilizing discontinuous sucrose density gradients and negative lectin selection. More than 99 +/- 1% (n = 4) of the isolated vesicles contain entrapped fluorescein dextran when analyzed by small-particle flow cytometry techniques. Similarly, flow cytometry analysis demonstrates brisk H(+)-adenosinetriphosphatase activity in virtually all the vesicles. Western blot analysis of the vesicle proteins with a polyclonal anticlathrin antibody stains bands consistent with clathrin and adaptins. When the isolated vesicles are decoated by exposure to 0.5 M tris(hydroxymethyl)aminomethane, the proteins released match the molecular weights of the proteins identified on Western blot analysis. Flow cytometry demonstration of brush border enzymes in > 99% of the vesicles and Western blot identification of maltase suggests both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Additionally, two glycoproteins detectable in this fraction on Western blot analysis and flow cytometry immunocytochemistry are derived from intermicrovillar clefts traffic into the endosomal pathway. Hence, apical proteins traffic into a population of CCV isolated from the rat renal cortex.
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PMID:Trafficking of apical proteins into clathrin-coated vesicles isolated from rat renal cortex. 818 87

In this study enzyme activities and lectin binding patterns in skeletal muscle from very old rats were investigated in order to evaluate changes in enzyme activity or carbohydrate expression in senile muscle. Activities for adenosine triphosphatase (ATPase), succinic dehydrogenase, non-specific esterase and the binding pattern for 31 lectins were investigated in the soleus muscles from very old (36 months) and young (3 months) rats. In ageing muscles atrophic, angulated muscle fibres are frequent. In cryostat sections these fibres were mostly but not always type II defined by the myosin ATPase reaction; few showed a strong esterase activity. Some showed strong activity for succinic dehydrogenase while others were weakly reacting. A number of lectins strongly bound to the sarcoplasm in angulated fibres while the binding to normal fibres in both old and young rat muscle was much weaker or even absent. Preferential binding to the ageing, angulated fibres was seen with Aleuria aurentia, Galantus nivalis, Caragana abborecens, Triticum vulgaris, Maackia amurensis, Sambucus nigra, Phaseolus vulgaris erythroagglutinin, and Phaseolus coccineus. Samples of homogenized and centrifuged muscles were run by electrophoresis and the gels blotted to nitrocellulose paper. Subsequent lectin staining of the blots detected that two glycoproteins with molecular weights around 25,000 and 21,000 daltons were present in old muscle, but not in young. Aberrant or elevated expression of sarcoplasmic glycoconjugates is involved in ageing muscle atrophy.
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PMID:Glycosylation pattern and enzyme activities in atrophic, angulated skeletal muscle fibres from ageing rats. 818 92

RCCT-28A cells, a continuous cell line of rabbit cortical collecting tubule origin, have been found to exhibit apical peanut-lectin binding, basal band-3 immunostaining and a transepithelial electrical resistance of 246 +/- 37 omega cm2. For the studies reported, confluent monolayers of RCCT-28A cells were grown on permeable wells and incubated in a control solution or in alkaline solutions by lowering PCO2. Equivalent H+ fluxes (JH+) into the apical solution (nmol.min-1.cm2) were measured in the absence of drugs and in the presence of: amiloride (A, 10(-3) M), N-ethylmaleimide (NEM, 5 mM) and omeprazole (OM, 100 microM) in the apical solution. After preincubation in control solutions JH+ was 21 +/- 2 while A had no effect. Addition of NEM diminished JH+ to 12 +/- 2 (P < 0.005), and OM diminished JH+ to 2 +/- 2 (P < 0.001 vs. control). Monolayers incubated at low PCO2 had a basal JH+ of 11 +/- 5. No effect on JH+ could be demonstrated under these conditions by addition of NEM or OM. Removal of K+ from the apical solution diminished apical acidification by 60%. The inhibitor of H+,K(+)-ATPase Schering 28080 (SCH) was tested at different concentrations and an inhibitory effect was demonstrated (JH+ -2 +/- 1 vs. 18 +/- 1, SCH vs. control, respectively). Probenecid and bafilomycin-A also decreased apical acidification and an apical base-equivalent extrusion was apparent under the inhibitors effect. JH+ was abolished by removal of Cl- from the basolateral solution.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of acid-base transport mechanisms in the kidney cell line RCCT-28A. 838 89

Cells from RCCT-28A cell line, which exhibited the highest peanut-lectin binding capacity [RCCT-28A(P+)] were separated and cultured attempting to obtain a population of HCO3- secreting cells. The mechanisms of apical solution acid or base extrusion were studied in confluent monolayers. In RCCT-28A (P+) cells net H+ flux (JH+, nmol.min-1.cm-2) was significantly lower (JH+ = 13 +/- 3) than that observed in the RCCT-28A cells (21 +/- 2). N-ethylmaleimide, Bafilomycin-A, omeprazole and Schering 28080 decreased JH+. Cl- removal from the basolateral side, in the presence or absence of SCH in the apical side, decreased JH+. Removal of apical Cl- abolished apical extrusion of base-equivalents. Incubation in a high bicarbonate solution results in base-equivalent flux to the apical side (JH+ = -7.0 +/- 1.0). Preincubation in a hyperosmotic medium (mannitol addition) at pH 7.5 decreased JH+ to -3.0 +/- 1.0. We conclude that, functionally, RCCT-28A(P+) cells are only quantitatively different from RCCT-28A cells. Acid secretion by these cells can be modulated by alkaline and by hyperosmolar preincubation and is most likely mediated by coexistent of apical H(+)-ATPase and H+,K(+)-ATPase. The main mechanism of net OH- secretion appears to be Cl-/HCO3- exchange at the apical side.
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PMID:Characterization of peanut-lectin (+) cells derived from the RCCT-28A cell line. 838 90

In summary, this review has provided information concerning the application of histochemical and cytochemical procedures used to detail the normal versus pathological cornea and ocular surface. Specifically, histochemical analysis has been used to study protein and peptide degradation in cornea, to analyze stromal non-collagenous and collagenous fibers and associated extracellular matrix. Cytochemistry of the ocular surface has been used to detail the morphology of corneal and conjunctival mucin. Use of small cationic probes as well as lectin-gold binding was advantageous to quantitatively demonstrate that ocular mucin contains sialylated residues and that the number of these residues significantly changes (increases) with age. These data are important in that the degree of sialylation has been shown to correlate with the ability of bacterial organisms to adhere to and infect the immature in contrast to the mature corneal surface. The use of lectin analysis of diseased ocular tissue also has shown that there are specific alterations in glycoconjugates which occur in the diseased versus normal human cornea. Wound healing in cornea is an important problem which has been studied at length using combined histochemical and biochemical approaches. Results support the hypothesis that apical cell surfaces of the leading edge of a migrating sheet differ from those of the normal epithelium. During wound healing, alpha 6 integrin expression by corneal epithelial cells has been demonstrated, but another protein, syndecan was only seen in non-migrating epithelium which had restratified. The association of immunoglobulins with the ocular surface epithelium of the cornea, their change with age and kinetics of appearance also has been demonstrated using a cytochemical approach. Histochemical procedures have been used to localize Class I and Class II molecules in cornea and conjunctiva. Class II antigen expression has been shown to be absent on corneal endothelium, but it can be induced by treatment with IFN-gamma. These data are of importance in corneal pathology such as that resulting in rejection of corneal transplants. Langerhans cells (Class II, Ia positive) also are not found in normal central cornea. They are localized in the peripheral cornea and are stained histochemically by ADPase, ATPase and by specific anti-Ia and other antisera. Increased numbers of LC have been demonstrated in cornea following various stimuli and in diseases of the cornea including both bacterial and viral induced keratitis.
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PMID:Corneal and ocular surface histochemistry. 845 77

The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation.
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PMID:Determinants of oligomeric structure in the chicken liver glycoprotein receptor. 850 42


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