Reconstitution of D-glucose transport in vesicles composed of lipid and a partially purified protein from the human erythrocyte membrane

Zala, Cedric A.; Kahlenberg, Arthur

doi:10.1016/s0006-291x(76)80212-4

Cited by 33 publications

(5 citation statements)

References 23 publications

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“…relative to L-glucose from sonicated liposomes, was measured by a slight modification of the method previously described (12). Briefly, the membrane protein fraction is incorporated into liposome bilayers by sonication with a suspension of erythrocyte lipids containing both D-[2-3 HI -and L-[ 1 -I4C] glucose, The sonicated suspension is then passed through a Bio-Gel P4 column which retards extraliposomal glucose, resulting in a concentration gradient between the inside and the outside of the liposomes.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of D‐glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane

Kahlenberg

Zala

1977

J Supramol Struct

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Elucidation of the mechanism of facilitated D-glucose transport in human erythrocytes is dependent on the identification and isolation of the membrane protein(s) mediating this process. Based on the fact that stereospecific D-glucose transport is reconstituted in liposomes prepared by sonication of a lipid suspension with ghosts or fractions derived from ghosts, a quantitative assay for the stereospecific D-glucose transport activity of these fractions was developed (Zala CA, Kahlenberg A: Biochem Biophys Res Commun 72:866, 1976). This assay was used to monitor the purification of ghosts. The solubilized membrane protein fraction was chromatographed on a column of diethylaminoethyl cellulose which was eluted stepwise with NaCl-phosphate buffers of increasing ionic strength. A fraction, eluted at an ionic strength of 0.1, displayed a 13- and 27-fold increase in reconstituted transport activity relative to ghosts and to the unfractionated Triton X-100 extract, respectively. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, consisted predominantly of the ghost proteins with an apparent molecular weight of 55,000, commonly designated as zone 4.5; periodic acid-Schiff-sensitive membrane glycoproteins 1-4 were absent. Transport reconstituted by this preparation of zone 4.5 membrane proteins was almost completely abolished by 1-fluoro-2,4-dinitrobenzene, mercuric chloride, and p-chloromercuribenzene sulfonate, but was unaffected by sodium iodoacetate. Extra- and intraliposomal phloretin and cytochalasin B, respectively, exhibited partial inhibition. The stereospecificity and inhibition characteristics of the reconstituted transport imply that all the components of the erythrocyte D-glucose transport system are contained in the zone 4.5 membrane protein preparation.

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Section: Methodsmentioning

confidence: 99%

Reconstitution of D‐glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane

Kahlenberg

Zala

1977

J Supramol Struct

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“…The types of compounds used have been substrate analogue covalent affinity labels (Taverna & Langdon, 1973b;Trosper & Levy, 1977), nonspecific covalent inhibitors such as maleimides (Batt et al, 1976) and fluorodinitrobenzene (Jung & Carlson, 1975), and a bound reversible inhibitor, cytochalasin B, in conjunction with differential membrane extraction and fluorodinitrobenzene labeling (Lienhard et al, 1977;Zoccoli et al, 1978; Jung & Rampal, 1977;Pinkovsky et al, 1978). The second general approach has been to extract, purify, and isolate a specific membrane protein which, when reassociated with a lipid bilayer, conferred upon it stereospecific glucose transport (Kasahara & Hinckle, 1976, 1977 Kahlenberg, 1976;Zala & Kahlenberg, 1976; Jones & Nickson, 1978; Goldin & Rhoden, 1978; Shanahan & Czech, 1977a,b;Phutrakul & Jones, 1979).…”

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Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 1. Inhibition of glucose transport

Mullins¹,

Langdon²

1980

Biochemistry

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Maltosyl isothiocyanate (MITC) has been synthesized from maltose with an overall yield of 88%. It has been found to be a potent irreversible inhibitor of zero trans influx of glucose with human erythrocytes. Kinetic analysis of glucose transport after treatment of erythrocytes with MITC revealed that VT was diminished while KT was unchanged. Transportable sugars and competitive inhibitors of monosaccharide transport protected against MITC inhibition, while carbohydrates which do not interact with the transporter gave no protection. Covalent inhibitors of anion transport were without effect on glucose transport. MITC fulfilled the kinetic requirements for an affinity label of the glucose transporter of human erythrocytes [Groman, E. V., Schultz, R. M., & Engel, L. L. (1977) Methods Enzymol. 46, 54].

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“…Kasahara & Hinkle (1976) as well as Zala & Kahlenberg (1976) and Kahlenberg (1976) initially reported that partial purification of Triton X-100 extracts of membranes or DMMA-extracted membranes yielded band 3 preparations which were active in reconstituting glucose transport in lipid vesicles and in glucose binding (Kahlenberg et al, 1971), while Zoccoli & Lienhard (1 977) concluded from differential extraction studies that bands I . 2.…”

Section: Discussionmentioning

confidence: 99%

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 2. Identification of the transporter

Mullins¹,

Langdon²

1980

Biochemistry

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Maltosyl isothiocyanate (MITC), a potent irreversible inhibitor of glucose transport in human erythrocytes [Mullins, R. E., & Langdon, R. G. (1980) Biochemistry (preceding paper in this issue)], has been found to react almost exclusively with band 3 of the human erythrocyte membrane. The incorporation of [14C]MITC into band 3 was found to be antagonized by transportable sugars or competitive inhibitors of transport. On the basis of [14C]MITC incorporation into band 3 and MITC inhibition of transport, it is estimated that there are 3 x 10(5) glucose transporters present in the erythrocyte membrane. It was found that [14C]MITC-labeled band 3 could be converted into 14C-labeled band 4.5 during the Triton X-100 extraction procedure described by Kasahara & Hinkle [Kasahara, M., & Hinkel, P. C. (1977) J. Biol. Chem. 252, 7384]. On the basis of the evidence presented here and in the preceding paper, it is suggested that in the native erythrocyte membrane a component of band 3 is the glucose transport protein and that during purification with nonionic detergents the transport protein may be enzymatically degraded with some retention of activity.

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Reconstitution of D-glucose transport in vesicles composed of lipid and a partially purified protein from the human erythrocyte membrane

Cited by 33 publications

References 23 publications

Reconstitution of D‐glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane

Reconstitution of D‐glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 1. Inhibition of glucose transport

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 2. Identification of the transporter

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