scholarly journals Multiple carbohydrate moieties on the Na+/H+ exchanger

1993 ◽  
Vol 289 (3) ◽  
pp. 637-640 ◽  
Author(s):  
R S Haworth ◽  
O Fröhlich ◽  
L Fliegel
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Terminal Region ◽  
Apparent Molecular Mass ◽  
Carbohydrate Moiety ◽  
Detectable Effect ◽  
Complex Type ◽  
Brush Border Membranes ◽  
Limited Digestion ◽  
Endoglycosidase H

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.

scholarly journals Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes

1988 ◽  
Vol 255 (1) ◽  
pp. 185-191 ◽  
Author(s):  
H Debiec ◽  
R Lorenc
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
High Specificity ◽  
Brush Border Membranes ◽  
Intestinal Brush Border

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

scholarly journals Characterization of the invertase from Pichia anomala

1995 ◽  
Vol 306 (1) ◽  
pp. 235-239 ◽  
Author(s):  
J Rodriguez ◽  
J A Perez ◽  
T Ruiz ◽  
L Rodriguez
Keyword(s):  
Carbon Source ◽  
Molecular Mass ◽  
Total Mass ◽  
Culture Medium ◽  
Thermal Sensitivity ◽  
Apparent Molecular Mass ◽  
Carbohydrate Moiety ◽  
Pichia Anomala

Synthesis of invertase (EC 3.2.1.26) in Pichia anomala is controlled by the carbon source in the culture medium. The enzyme was purified to homogeneity from P. anomala cells fully derepressed for invertase synthesis and shown to be a multimeric glycoprotein composed of identical subunits with an apparent molecular mass of 86.5 kDa. The carbohydrate moiety accounts for approx. 30% of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the polypeptide. Most of the characteristics of the enzyme analysed in this study were similar to those previously reported for other yeast invertases, with the remarkable exception of its thermal sensitivity which appears after 15 min incubation at temperatures above 32 degrees C.

scholarly journals Human small intestinal angiotensin-converting enzyme: intracellular transport, secretion and glycosylation

1993 ◽  
Vol 296 (3) ◽  
pp. 607-615 ◽  
Author(s):  
H Y Naim
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Molecular Mass ◽  
Brush Border ◽  
Intracellular Transport ◽  
Apparent Molecular Mass ◽  
Intestinal Cells ◽  
Converting Enzyme ◽  
Human Biopsy ◽  
Membrane Bound ◽  
Small Intestinal

Human intestinal angiotensin-converting enzyme (ACE) exists in the brush-border membrane as a monomeric protein of apparent molecular mass 184 kDa. It is associated with the membrane via a hydrophobic segment and has a transmembrane orientation [Naim (1992) Biochem. J. 286, 451-457]. In addition to the membrane-bound form (ACEm), hydrophilic forms of ACE (ACEsec) can be identified in biosynthetically labelled intestinal cells. Thus the culture medium of biosynthetically labelled human biopsy samples contains an ACE molecule which has an apparent molecular mass similar to that of its membrane-bound counterpart. The secreted ACEsec forms follow a precursor/product relationship with the mature ACE molecule. The effect of the monomeric structure of ACE in its intracellular transport and secretion was investigated by pulse-chase experiments on human biopsy samples labelled with [35S]methionine. The results reveal 2-3-fold slower transport of ACE from the endoplasmic reticulum (ER) to the Golgi as compared with the homodimeric proteins dipeptidylpeptidase IV and aminopeptidase N. Further, the transport kinetics of ACE are comparable with those of human sucrase-isomaltase and human maltase-glucoamylase, two brush-border disaccharidases that do not form homodimers in the ER of human small-intestinal cells. These findings strongly suggest that homodimerization of brush-border proteins may influence the rate of transport of these proteins from the ER to the Golgi. The effect of glycosylation on the transport and secretion of ACE was investigated by utilizing several inhibitors of glycan processing. Here, secretion of ACE molecules continued to take place, albeit to a considerably lesser extent. In fact, approx. 2-fold less ACE molecules were secreted in the presence of inhibitors of ER glucosidases I and II and cis-Golgi mannosidase-I, suggesting that carbohydrate processing is important in the attainment of a transport-competent conformation.

scholarly journals Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes

1992 ◽  
Vol 286 (1) ◽  
pp. 97-102 ◽  
Author(s):  
H Debiec ◽  
R Lorenc ◽  
P M Ronco
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Broad Band ◽  
Gel Filtration ◽  
Phosphate Transport ◽  
Rabbit Kidney ◽  
Dilution Method ◽  
Native Form ◽  
Brush Border Membranes ◽  
Transporter Protein

A protein with Na+/Pi co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20%), phosphatidylcholine (20%), phosphatidylethanolamine (30%) and phosphatidylserine (30%) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/10 s per mg) than with a K+ gradient (65 pmol/10 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value (‘overshoot’) when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na(+)-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170 +/- 10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/Pi co-transporter from rabbit kidney brush-border membranes.

scholarly journals Identification and characterization of the major stilbene-disulphonate- and concanavalin A-binding protein of the porcine renal brush-border membrane as aminopeptidase N

1990 ◽  
Vol 271 (1) ◽  
pp. 147-155 ◽  
Author(s):  
H See ◽  
R A F Reithmeier
Keyword(s):  
Molecular Mass ◽  
Concanavalin A ◽  
Brush Border ◽  
Membrane Fraction ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Aminopeptidase N ◽  
Brush Border Membranes ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

A 130 kDa glycoprotein (GP 130) was purified from porcine renal brush-border membranes by affinity chromatography using immobilized 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate (SITS)- and concanavalin A-Sepharose. GP 130 was the major concanavalin A-binding protein in porcine renal brush-border membranes and also bound Ricinus communis (castor-bean) and wheat-germ agglutinins. Endo-beta-N-acetylglucosaminidase F reduced the molecular mass of GP 130 by 20 kDa as determined by SDS/PAGE, whereas endo-beta-N-acetylglucosaminidase H reduced the molecular mass by 5 kDa, showing that GP 130 contained both complex and high-mannose carbohydrate structures. Western-blot analyses using an antibody raised against GP 130 showed that it was localized to the brush-border membrane fraction and was present in a membrane fraction of the pig kidney cell line LLC-PK1. The N-terminal sequence and amino acid composition of GP 130 showed that GP 130 is similar to rat kidney zinc peptidase and human intestinal aminopeptidase N. GP 130 had aminopeptidase N enzymic activity and was inhibited by bestatin (Ki = 36 microM), 1,10-phenanthroline (Ki 30 microM), Zn2+ (Ki 26 microM), Cu2+ (Ki 260 microM), pre-incubation with EDTA and by a polyclonal antibody against GP 130. Bicarbonate and iodide blocked the binding of GP 130 to the SITS-affinity resin, showing that GP 130 has an anion-binding site. Neither these anions nor stilbene disulphonates affected the aminopeptidase N activity of GP 130.

scholarly journals PHOSPHATE (Pi) TRANSPORT IN RENAL BRUSH BORDER MEMBRANES (BBMV) IN NEWBORN PUPPIES (P)

1984 ◽  
Vol 18 ◽  
pp. 366A-366A
Author(s):  
Eddie S Moore ◽  
Eunice G John ◽  
Lawrence Rufr ◽  
Christine S Mooers ◽  
Nochik Park ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membranes ◽  
Pi Transport ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border
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