Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril

Ma, Markwell; Cf, Fox

doi:10.1021/bi00615a031

Cited by 877 publications

(330 citation statements)

References 35 publications

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“…Purified antibodies were iodinated using an iodination Reagent (IODO-GEN, Pierce Chemical Co., Rockford, ILL 1,3,4,6-tetrachloro 3¢t 6a-diphenyl glycoluril from Pierce Chemical Co. (Markwell and Fox, 1978). Competitive RIAs were done by modification of the methods of Kiehart et al (1986) and Peltz et al (1985).…”

Section: Competitive Binding Radioimmunoassay (Ria )mentioning

confidence: 99%

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

Ingold

Cohn

Scholey

1988

The Journal of Cell Biology

174

153

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Abstract, We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesininduced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.V ARIOUS forms of intracellular transport are thought to depend upon microtubule-associated mechanochemical ATPases (Vale, 1987). One such enzyme, kinesin (Vale et al., 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al., 1985a, b;Scholey et al., 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al., 1987;Cohn et al., 1987;Saxton et al., 1988;Gelles et al., 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al., 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al., 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al., 1988). Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al., 1988;Bloom et al., 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al., 1986;Vale, 1987;Leslie et al., 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.Antibodies that inhibit mechanochemical activity have been extremely useful in probing the functions of myosin and 1. Abbreviations used in this paper: MT(s), microtubule(s); PMEG, 0.9 M glycerol, 0.1 M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 100 Ixg/ml soybean trypsin inhibitor, 1 mg/mlp-tosyl-Larginine methyl ester hydrochloride, 10 Ixg/ml leupeptin, pepstatin, and aprotinin; RT, room temperature. dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;O...

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Section: Competitive Binding Radioimmunoassay (Ria )mentioning

confidence: 99%

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

Ingold

Cohn

Scholey

1988

The Journal of Cell Biology

174

153

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“…FN and type IV collagen were labelled with carrier-free Na1251 (Amersham, Buckinghamshire, England and New England Nuclear, Boston, MA, USA) using the Iodogen [30] (Pierce, Rockford, USA) and the chloramine T methods [31]. The specific activity obtained ranged from 2.5 to 3.6/~Ci//zg of protein.…”

Section: Binding Of Radiolabelled Glycoproteins To Bacteriamentioning

confidence: 99%

A novel lectin‐independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

et al. 1989

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Binding of P fimbriae of uropathogenic Escherichia coil to purified human plasma flbronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized flbronectin was not inhibited by ,,-D-GaI-( 1-4)-fl-D-Gal-1-O-Me, a receptor analog of P funbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble ~'5I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the flmbriae.

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“…Reaction vessels were prepared by coating borosilicate glass tubes (10 x 75 mm) with 10 #g of this reagent as previously described (28,29). 1 nmol of either RC, LM, or H in 100 #1 of PBS, 0.1% LDAO, or 2 nmol of L or M in 100 t.d of PBS.…”

Section: Radioiodination Of Rc Polypeptidesmentioning

confidence: 99%

Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

Valkirs¹,

Fehér²

1982

The Journal of Cell Biology

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The localization of the reaction center polypeptides (L, M, and H) in the membranes of both the wild-type, strain 2.4.1, and the carotenoidless mutant, R-26, of Rhodopseudomonas sphaeroides was determined by using affinity-purified antibodies specific for these proteins.Binding of the antibodies to reaction center subunits in spheroplasts was visualized in the electron microscope by immunoferritin labeling. The H and M subunits were labeled at both the cytoplasmic and the periplasmic surfaces of the membrane, whereas the,L subunit was labeled only at the periplasmic surface of the membrane. Thus, the reaction center is asymmetrically oriented in the membrane with at least two subunits (H and M) spanning the membrane.The plasma membrane of the photosynthetic bacterium, Rhodopseudomonas sphaeroides, exhibits an intricate series of invaginations (1, 2) that harbor the reaction center (RC) polypeptide subunits. The RCs have been isolated by detergent extraction (for a review, see, for example, reference 3) and are believed to be integral membrane proteins (4). The subunits, designated L, M, and H, are in a 1:1:1 stoichiometry (5). Their molecular weights, determined from an analysis of their amino acid compositions (6, 7) were found to be 28,000, 32,000, and ~34,000, respectively. Each RC contains four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones, and one iron (3). Together with these cofactors, the RC protein accomplishes the conversion of light into electrochemical energy.According to the chemiosmotic hypothesis (8), the topographical organization of the membrane components is fundamental to the directional transfer of protons and electrons and to the coupling of these events to the generation of ATP. Thus, localization of the RC subunits with respect to the membrane should aid in determining their function in the primary charge separation which initiates cyclic electron transfer.Several techniques have been used to investigate the topography of the RC subunits. They include: precipitation with (9), and adsorption of (10), antisera, labeling with antibodies (11-14), radiochemical labeling ( 15-18), photoaffinity labeling (19), and enzymatic digestions (16,18,20). These studies have shown that the RC is an integral membrane protein with the H subunit being exposed on the cytoplasmic side of the membrane. However, no clear concensus has been reached concerning the topography of the other subunits (for a more detailed discussion, see last section of this paper).We used specific antibodies to probe the topography of RCs in the membrane by indirect immunoferritin labeling (21). In this technique, the photosynthetic membrane is first exposed to rabbit antibodies directed against the RC subunits and then to ferritin-conjugated goat antibodies that bind to rabbit IgG. Ferritin is an electron-dense molecule, thereby permitting localization of the binding site by electron microscopy.We performed our initial work on chromatophores, which are closed, inverted membrane vesicles purified from disrupte...

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Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril

Cited by 877 publications

References 35 publications

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

A novel lectin‐independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

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