Binding of the nonionic detergent [3HrTriton X-100 by diphtheria toxin, by the nontoxic serologically related protein crossreacting material (CRM) 45, and by their respective A and B fragments has been studied. If Fragment A then catalyzes the transfer of the ADP-ribosyl group from NAD+ to elongation factor 2 (EF-2), thereby causing its inactivation (1, 2).Previous studies (3, 4) with mammalian cell cultures have shown that each sensitive cell carries about 4000 specific surface membrane receptors that initially react reversibly with groups located near the COOH-terminus of the toxin B fragment. This initial rapid reaction is followed by a slow irreversible process involving a major conformational change, during which the molecule enters the plasma membrane. Finally, fragment A is split off to reach the cytoplasm while B apparently remains behind in the membrane. If this model is indeed correct, we would expect that fragment B should behave like other membrane proteins and should contain a hydrophobic "domain" (5) which becomes inserted into the lipid bilayer during the entry process.Membrane proteins may be extracted into aqueous solvents that contain a nonionic detergent such as Lubrol or Triton X-100. During this process, protein-bound phospholipid molecules are replaced by molecules of the detergent. By measuring the quantity of detergent that it can bind, it is possible to estimate the fraction of a protein molecule's surface capable of hydrophobic interaction (6, 7). We are now reporting studies on the binding-of Triton X-100 to diphtheria toxin, to its A and B fragments, and to the nontoxic, serologically related tox gene product, crossreacting material (CRM) 45, which lacks the 17,000 dalton COOH-terminal amino acid sequence of the intact toxin molecule (8). Based on our findings, we are proposing a model to describe the process by which the diphtheria toxin A fragment is transported across the plasma membrane. MATERIALS AND METHODSDetergent Preparation. Triton X-100 (polyoxyethylene octyl phenol, averaging 9.6 ethylene oxide units per monomer) was obtained from Rohm & Haas Co., Philadelphia. An aqueous solution of ring-labeled [3H]Triton X-100 (0.93 mg/ml) was a gift from Steven Clarke. The concentration of this stock solution was calculated from the absorbance at 274 nm of dilutions in 1 mM Tris-HCI buffer containing 0.1 M Na2SO4 at pH 7.5, assuming A (1%) = 23.2. The specific activity was 125 cpm/,gg of [3H]Triton X-100 as determined in 3 ml of Aquasol (New England Nuclear) in a Beckman LS230 liquid scintillation counter. The critical micelle concentration (CMG) of Triton X-100 was determined using methyl orange by the method of Benzonana (9) to be 0.13 mg/ml in the 10 mM phosphate buffer at pH 7.2 used for binding studies. Labile tritium was estimated by passing a small volume of the stock [3H]Triton X-100 solution through a Sephadex G-0 Icolumn. Almost 99% of the radioactivity emerged in the void volume.Proteins. Partially purified diphtheria toxin (30-5% nicked) was obtained from Connaugh...

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.