The use of the iodinating reagent 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (chloroglycoluril) to selectively label membrane surface proteins was investigated with the following systems: enveloped viruses (Sendai and Newcastle disease viruses), human erythrocytes, and nucleated cells propagated both in suspension (EL-4) and in monolayer culture (BHK-21). Conditions are described for specifically iodinating surface proteins while maintaining full virus integrity or cell viability. Comparison of the chloroglycoluril method with the lactoperoxidase and chloramine-T methods for labeling surface membrane proteins shows that the chloroglycoluril method has a number of advantages: It routinely produces a 3- to 17-fold greater specific radioactivity without sacrificing viral or cellular integrity, it is technically simpler to use, it does not require the addition of extraneous protein to initiate the reaction nor a strong reducing reagent to terminate it. Chloroglycoluril also proved to be an effective substitute for chloramine-T in the nonvectorial labeling of viral and cellular proteins. Membrane protein samples were solubilized with the detergent sodium dodecyl sulfate before iodination or labeled in the presence of high iodide concentrations without prior solubilization. The resulting specific radioactivities generated by the use of chloroglycoluril were equal to or greater than those generated by the chloramine-T method. The effectiveness, simplicity of use, and versatility of chloroglycoluril recommend it as an iodinating reagent for both surface-specific and nonvectorial labeling of membrane systems.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.

Arrhenius plots describing the logarithm of the rate of $-galactoside transport cs. the reciprocal of the absolute temperature of transport assay are biphasic in slope with intersects (transport transition temperatures) at 30 and 13" for cells of an Escherichia coli unsaturated fatty acid auxotroph grown at 37" in medium supplemented with elaidic acid and oleic acid, respectively. When transport is induced at 37" for a short time period (less than one-eighth of a generation of growth) after shifting from growth with an oleic acid supplement to growth with an elaidic acid supplement or vice versa, a single transition temperature for @-galactoside transport is detected in each case, and the temperatures at which the transitions are observed indicate that transport is primarily influenced by the average fatty acid composition of the membrane lipids. If transport induction proceeds at 25" after the fatty acid shift, however, the Arrhenius plots are triphasic. The two observed transport transition temperatures reflect b e v e r a l independent studies point to an interrelationship between protein and lipid biosynthesis during the morphogenesis of certain microbial transport systems. Following a period of starvation for an essential fatty acid, induction of a fully functional lactose transport system in unsaturated fatty acid auxotrophs of Escherichia coli is blocked, even though the induction of @galactosidase and thiogalactoside transacetylase proceeds normally (Fox, 1969;Overath et al., 1971a). Addition of an essential fatty acid subsequent to the starvation period does not result in a recovery of transport activity (Fox, 1969). Induction of functional lactose transport system activity

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