The insulin receptor has been shown to be a protein kinase which phosphorylates its substrates on tyrosine residues. To examine the acceptor specificity of affinity-purified insulin receptor/kinase, hydroxyamino acid containing analogues of the synthetic peptide substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly were prepared. Substitution of serine, threonine, or D-tyrosine for L-tyrosine completely ablated the acceptor activity of the synthetic peptides. These peptides, along with a phenylalanine-containing analogue, did serve as competitive inhibitors of the insulin receptor/kinase with apparent Ki values in the range of 2-4 mM. These data suggest that the insulin receptor/kinase is specific for tyrosine residues in its acceptor substrate and imply that serine phosphate or threonine phosphate present in receptor is due to phosphorylation by other protein kinases. The kinetics of the phosphorylation of the L-tyrosine-containing peptide were examined by using prephosphorylated insulin receptor/kinase. Prephosphorylation of the receptor was necessary to maximally activate the kinase and to linearize the initial velocity of the peptide phosphorylation reaction. The data obtained rule out a ping-pong mechanism and are consistent with a random-order rapid-equilibrium mechanism for the phosphorylation of this peptide substrate. Additional experiments demonstrated that the autophosphorylated insulin receptor was not able to transfer the preincorporated phosphate to the synthetic peptide substrate. Thus, the insulin receptor/kinase catalyzes the reaction via a mechanism that does not involve transfer of phosphate from a phosphotyrosine-containing enzyme intermediate.

Effects of isocaloric changes in dietary fat on plasma lipoproteins and lipids and enzymes of erythrocytes and leucocytes were assessed. Subjects with a higher Brocca index showed increase in total and LDL cholesterol, significant reduction in HDL cholesterol, and increased total cholesterol:HDL cholesterol ratio after high-fat diet consumption. Due to high-fat diet feeding, erythrocyte membrane and leucocyte cholesterol and phospholipid contents were increased, cholesterol:phospholipid molar ratio was elevated, and erythrocyte enzymes (G6PD and 6PGD) and leucocyte enzymes (CEH and CES) were elevated. Erythrocyte membrane glycoprotein components showed marked increase, indicating possible alterations of membrane surfaces. The metabolic alterations were reversed slowly after resumption of the normal (low-fat) diet. Body weight plays an important role in the alterations in major lipoprotein cholesterol contents in response to changes in dietary fat composition. Cellular changes indicate alterations in structure and function of blood cells due to high-fat diet feeding.

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