Functional significance of cysteine residues in the delta opioid receptor studied by site-directed mutagenesis.

Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore, these results support the idea that the striking effects of sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.

[Indicators of lipid peroxidation in the blood in hereditary predisposition to arteriosclerosis]. / Pokazateli perekisnogo okisleniia lipidov krovi pri nasledstvennom predraspolozhenii k aterosklerozu.

In members of the families whose parents had atherosclerosis complicated by macrofocal myocardial infarction or stroke, the serum level of lipid peroxidation products was correlated to enzymatic activity of neutrophil and red blood cells oxidation-antioxidation. In persons with hereditary predisposition to atherosclerosis both with clinical signs of atherosclerosis and phenotypically healthy against the control group there was elevated content of plasma acylhydroperoxides and hypoactivity of neutrophil myeloperoxidase. Determination of lipid peroxidation products by malonic dealdehyde showed this parameter to be higher in members of the families of the study group and in those with cardiovascular disorders. For those whose parents had atherosclerosis versus control subjects there were no differences in the activity of superoxide dismutase, glutation peroxidase and catalase in the blood red cells. Shifts in lipid peroxidation and activity of blood myeloperoxidase are identical in type and may represent a pathogenetic ling in formation of hereditary predisposition to cardiovascular disorders of atherosclerotic origin, the detection of which becomes feasible in a subclinical period.

[Blood lipids and lipid peroxides in the families of patients with ischemic heart disease]. / Lipidy i lipoperoksidy krovi v se'miakh bo'lnykh ishemicheskoi bolez'niu serdtsa.

Levels of some fractions of blood neutral lipids and lipoperoxides were compared in the families of patients with coronary heart disease. The patients and their relatives (including healthy ones) were found to show a statistically significant increase in the atherogenic index, total cholesterol, low density lipoprotein cholesterol, triglyceride, total lipid, and lipoperoxide concentrations and a decrease in high density lipoprotein cholesterol levels as against controls. The changes in the values of lipids and lipoperoxides in the families of patients with coronary heart disease were homogenous and might be one of the pathogenetic links in the formation of hereditary predisposition to atherosclerotic cardiovascular diseases which might be detected in a prehospital period.