Radiation Inactivation of Glutamate Dehydrogenase Hexamer: Lack of Energy Transfer Between Subunits

Kempner, Ellis S.; Miller, J.H.

doi:10.1126/science.6635656

Cited by 56 publications

(29 citation statements)

References 18 publications

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“…However, because only a single protein species (5, 6) is required for transport activity (22), our results indicate that the function of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical 73-kDa subunits arranged as a homotetramer. This model is similar to that proposed in a radiation inactivation study of the six independent subunits of glutamate dehydrogenase (14).…”

Section: Discussionsupporting

confidence: 78%

“…When a highenergy electron hits a polypeptide chain, the deposited energy completely destroys the chain, resulting in loss of function. The energy can be absorbed by a single polypeptide and not spread to adjacent chains (14); in such oligomeric proteins, each subunit is destroyed as a single entity.…”

Section: Discussionmentioning

confidence: 99%

“…The computer regression program was constrained such that J/Jo = 1 when D = 0. The molecular mass of the functional unit, or target size, was subsequently determined (13)(14)(15) from the relationship: target mass = 17.9 x 105 k, [1] which includes the temperature correction factor for irradiations performed at -135°C. Dosimetry was performed as described elsewhere (16 (5).…”

Section: Methodsmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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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“…These results are not unexpected because the S-S bond is chemically similar to C-C and C-N bonds in proteins, which have previously been shown to conduct destructive energy (2)(3)(4)13). The radiation sensitivity of disutfide bonds should be a chemical property of the bond rather than a property of the, secondary or tertiary structure of the polymer.…”

Section: Discussionmentioning

confidence: 66%

Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge.

Haigler¹,

Woodbury²,

Kempner³

1985

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

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The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfilde bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.Ionizing radiation damages molecular structure. Chemical bonds in macromolecules are broken and new ones form, leading to changes in physical, chemical, and biological properties. In a solid, changes in irradiated matter are due to the direct action of radiation on the molecules in question. The initial interaction is a primary ionization of an orbital electron in which large amounts of energy [about 1500 kcal/mol (1 kcal = 4.18 kJ)J are transferred to macromolecules. The subsequent events are only partially understood, but they clearly involve the transfer ofenergy to other regions of the molecule. In addition to the theoretical interest in these processes, this subject has attracted considerable research because high-energy radiation inactivation can be used-to measure the functional size of biologically active macromol'-ecules (see refs. 1 and 2 for reviews). The method is conceptually simple: if a polypeptide chain is "hit" it is completely inactivated; if a "hit" does not occur on a polypeptide it is not affected. By exposing biologically active molecules to increasing doses of radiation and measuring the surviving activity, the functional size of the structure(s) responsible for the activity can be determined by target analysis.The effects of ionizing radiation depend on the structural requirements for transfer of the energy deposited by a primary ionization. Evidence has been presented that radiation damage in synthetic polymers occurs at considerable distance from the primary ionization (3, 4) and throughout a polypeptide chain no matter where the original hit occurs (5). These data provide no insight into the radiation sensitivity of disulfide bonds, which often play a critical role in the structure and function ofproteins. In this report the disulfidelinked heterodimeric protein ricin was studied as a model to determine the ability of disulfide bonds to transmit destructive energy.Ricin is a toxic plant lectin that consists of two polypeptide chains, A and B, of known amino acid sequence (6, 7) that are joined by a single disulfide bond (Fig. 1) eukaryotic ribosomes. The enzymatic assay for A chain activity was used to determine if destructive energy could be transferred to the A chain after a primary ionization on the B chain. Iftransfer occurs, irradiation ofintact ricin should give a target size corresponding to the molecular size of dimeric ricin, but if the A and B chains are energetically isolated with regard to ionizing radiation, th...

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“…However, information about functional molecular size and structure of the native co-transporter is lacking. Radiationinactivation analysis with high-energy electron radiation is a method permitting the measurement of the size of functional units in situ without the need for isolating the protein components of the transporter, which might alter its subunit assembly or functional properties [28][29][30]. This methodology has been successfully applied to various transport systems such as the Na+/Dglucose co-transporter [31][32][33][34][35], the multispecific hepatic bile acid transporters [36][37][38], the Na+/phosphate transporter from kidney [34], or the p-aminohippurate transporter in the basolateral membrane of renal proximal tubules [39].…”

Section: Introductionmentioning

confidence: 99%

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 of Glutamate Dehydrogenase Hexamer: Lack of Energy Transfer Between Subunits

Cited by 56 publications

References 18 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 of ricin occurs with transfer of destructive energy across a disulfide bridge.

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

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