Radiation inactivation analysis of kidney microvillar peptidases

Fulcher, I S; Ingram, Jean C.; Kenny, A J

doi:10.1016/0014-5793(86)80921-8

Cited by 7 publications

(3 citation statements)

References 20 publications

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“…The theory and practice of radiation inactivation is well established in confirming the functional unit size of a wide variety of enzymes (11). The technique has also been used to determine the in situ functional unit size of brush border membrane-bound enzymes (24,25) and transporters (9,10). Specifically, the radiation target size of the kidney brush border membrane Na+/ glucose cotransporter in situ has been measured in a variety of species, with a target size of 288 kDa (9) and 298 kDa (26) for transporter activity.…”

Section: Discussionmentioning

confidence: 99%

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Stevens

Fernandez

Hirayama

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-1350C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10-'9 J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na'-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na'-dependent cotransporter activity JfaX without affecting apparent Km. The size of the transporting functional unit was 290 ± 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 ± 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.The intestinal brush border membrane Na+/glucose cotransporter is among the most thoroughly studied eukaryotic systems demonstrating secondary active (ion-coupled) transport. Biochemical and immunological experiments have identified the cotransporter as a polypeptide of Mr 70,000-75,000 on SDS/PAGE reducing gels (1-3), and the cloned cotransporter has a predicted molecular mass of 73 kDa (5, 6). However, information regarding structure-function relationships of the cotransporter in situ in the native membrane is lacking. In this study we used high-energy electron radiation inactivation to investigate the membrane-bound in situ size of the cotransporter. Our results indicate that the cotransporter functions in the membrane as a 290-kDa homotetramer comprised of four independent 73-kDa subunits. METHODS AND MATERIALSBrush border membrane vesicles were prepared from male New Zealand White rabbit small intestines by a 10 mM MgCl2 precipitation procedure (7,8). The membrane vesicles were enriched =15-fold in the brush border membrane marker enzymes alkaline phosphatase, leucine aminopeptidase, sucrase, and 'y-glutamyltranspeptidase. Vesicles (20 ,ug of protein per ,l) were equilibrated in a cryoprotectant buffer (9, 10) that contained 14% (vol/vol) glycerol, 1.4% (wt/vol) D-sorbitol, 150 mM KCI, and 5 mM Hepes-Tris (pH 7.5). Beliveau et al. (9, 10) have established that this buffer preserves glucose and phosphate transport, and Na+/H+ antiport in kidney brush border membrane vesicles. Aliquots of the vesicle suspension were frozen in 2-ml glass ampules (type 12012; Kimble, Toledo, OH). The sealed ampules were then kept at -80'C and transported on dry ice. The membranes were irradiated at -1350C with a beam of 13 MeV (1 eV = 1.602 x 10-1' J) electrons produced by a linear accelerator, as described elsewhere (11). After irradiation the ampules were held at -800C until...

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Section: Discussionmentioning

confidence: 99%

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Stevens

Fernandez

Hirayama

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…The main advantage of the radiationinactivation method is that it permits the measurement of the size of functional units in situ, without the need for isolating the protein, which might alter its subunit assembly or functional properties. This method has recently been applied to the brush-border-membrane vesicles to study peptidases [9] and other membranebound enzymes [10]. There are, however, very few studies concerning its applicability to transporters from brushborder membranes.…”

Section: Introductionmentioning

confidence: 99%

Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers

Béliveau¹,

Demeule²,

Ibnoul-Khatib³

et al. 1988

Biochemical Journal

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Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5'-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.

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“…These values clearly underestimate the real molecular size of the polypeptide chains of the respective enzymes. Radiation analysis of kidney microvillar peptidases of freezedried proteins, however, revealed target-size molecular masses very similar to the accurate molecular masses of the purified enzymes [54]. In our hands, the molecular mass ofaminopeptidase N was determined as 101 +29 kDa and that of sucrase as uptake 19 These experiments revealed that our experimental approach should allow the determination of the accurate functional molecular mass of the Na+/bile acid co-transport system.…”

Section: Functional Molecular Mass Of the Na+-dependent [3h]taurocholmentioning

confidence: 77%

Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum

Kramer

Girbig

Gutjahr

et al. 1995

Biochemical Journal

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The functional-unit molecular size of the Na+/bile acid cotransport system and the apparent target size of the bile-acid-binding proteins in brush-border membrane vesicles from rabbit ileum were determined by radiation inactivation with high-energy electrons. The size of the functional transporting unit for Na(+)-dependent taurocholate uptake was determined to 451 +/- 35 kDa, whereas an apparent molecular mass of 434 +/- 39 kDa was measured for the Na(+)-dependent D-glucose transport system. Proteins of 93 kDa and 14 kDa were identified as putative protein components of the ileal Na+/bile acid cotransporter in the rabbit ileum, whereas a protein of 87 kDa may be involved in passive intestinal bile acid uptake. Photoaffinity labelling with 3- and 7-azi-derivatives of taurocholate revealed a target size of 229 +/- 10 kDa for the 93 kDa protein, and 132 +/- 23 kDa for the 14 kDa protein. These findings indicate that the ileal Na+/bile acid co-transport system is in its functional state a protein complex composed of several subunits. The functional molecular sizes for Na(+)-dependent transport activity and the bile-acid-binding proteins suggest that the Na+/bile acid co-transporter from rabbit ileum is a homotetramer (AB)4 composed of four AB subunits, where A represents the integral 93 kDa and B the peripheral 14 kDa brush-border membrane protein.

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Radiation inactivation analysis of kidney microvillar peptidases

Cited by 7 publications

References 20 publications

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers

Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum

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