Mary Ann K. Markwell

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.

Abstract Liver and kidney organelles from rat and pig were separated by isopycnic sucrose density gradient centrifugation and located by marker enzymes. Carnitine palmitoyltransferase was shown to be exclusively a mitochondrial enzyme. In liver, approximately 52% of carnitine acetyltransferase activity was mitochondrial, 14% peroxisomal, and 34% located in a lipid-rich membranous fraction. Microsomes were a component of this last fraction and, when isolated by differential centrifugation, contained carnitine acetyltransferase activity. This enzyme has not previously been reported to be in peroxisomes. The specific activity of carnitine acetyltransferase in liver peroxisomes was two to three times greater than in the mitochondria or microsomes. Partial fractionation of broken rat liver peroxisomes into core, membranes, and the soluble matrix indicated that carnitine acetyltransferase had a similar distribution to the matrix enzyme, catalase. In gradients of rat and pig kidney, carnitine acetyltransferase was found primarily in the mitochondrial fractions. This enzyme was also not detected in microbodies, mitochondria, or microsomes from plants. Carnitine acetyltransferase activity in liver fractions was confirmed by three separate assays—an 1(-)-carnitine-dependent release of coenzyme A (CoA) from acetyl-CoA, identification of the 14C-labeled reaction product acetylcarnitine, and the 1(-)-acetylcarnitine-dependent formation of acetyl-CoA from CoA. Carnitine acyltransferase activity for octanoyl-CoA in hepatic peroxisomes and microsomes was about equal to activity for acetyl-CoA. In the mitochondria, activity for octanoyl-CoA was six times greater than for acetyl-CoA.