Sterol Binding Sites Research Articles

Evidence for a reactive sulfhydryl in the DNA binding domain of the 1,25-dihydroxyvitamin D 3 receptor

Evidence is presented here that organomercurial binding to a reactive sulfhydryl group is capable of altering the DNA-binding characteristics of the 1,25-dihydroxyvitamin D receptor (D-receptor). Accordingly, hormone-free receptor (R o) binding to DNA-cellulose is inhibited in a concentration-dependent fashion with both HgCl 2 and p-chloromercuribenzene sulfonate (pCMBS) with complete inhibition evident at 1.0 mM. Further, low concentrations (0.5 mM) of mercurials are also capable of dissociating preformed DNA-receptor complexes, a process reversible with excess thiol reagent such as monothioglycerol. These findings are in contrast to alkylating reagents such as iodoacetamide, which is capable of only partially inhibiting the formation of the receptor-DNA duplex (37% at 25 mM). Once created, however, the duplex is completely insensitive to dissociation (even at 25 mM). These results imply that in addition to the association of a cysteine(s) moiety in or near the sterol binding site, modification of a similarly reactive group(s) can also alter the D-receptor's DNA-binding domain.

Release of phagocytosed cholesterol by liver macrophages and spleen cells

The release of radioactive cholesterol from spleen cells and liver macrophages isolated after the intravenous injection of labeled colloidal cholesterol suspensions was studied. The cells actively oxidized [I- 14C]glucose and synthesized various phospholipids and protein when incubated in albumin-containing media, but released very little labeled cholesterol. Addition of serum or serum lipoproteins to the medium increased the release of labeled cholesterol severalfold, whereas the addition of lipoprotein-free serum had no effect. Once serum lipoproteins were present the release of labeled cholesterol was not affected by the addition of cycloheximide, NaF or KCN, or by the addition of physiological concentrations of lysolecithin and linoleic acid. Depletion of serum unesteriued cholesterol by the action of lecithin cholesterol acyltransferase did not alter the release of ingested labeled cholesterol to the incubation medium. The studies indicate that reticuloendothelial cells do not secrete cholesterol. Yet, once the phagocytosed cholesterol has been solubilized, it equilibrates rapidly with the cholesterol of the serum lipoproteins in the medium. It is suggested that phagocytic cells dispose of ingested cholesterol by a continuous exchange of the sterol between cell membranes and soluble lipoproteins and that the rate of net sterol transfer is determined by the relative degree of saturation of intracellular and extracellular sterol binding sites on membranes and soluble lipoproteins.

Binding of antibiotics to tissue homogenates.

Binding of metabolites and drugs, including antibiotics, to serum proteins has been extensively studied. This phenomenon has important biologic and therapeutic significance [1-3]. The ability of tissues to bind has also been explored, particularly with organic dyes and biologically active agents. In some instances, binding of drugs to specific sites in microorganisms can be linked closely with their mode of action. For example, the polymyxins owe their biologic activity to binding to polyphosphate groups of phospholipids in the bacterial cell membrane [4], and amphotericin B requires sterol binding sites in fungal cells [5]. Antitrypanosomal activity of acriflavine has been shown to depend upon binding of the drug to the parasite [6]. The receptor site theories of pharmacology and virology depend largely on evidence of specific combination between drug or virus and complementary structures on cell membranes. Some of these concepts of drug action were clearly visualized by Ehrlich and Morgenroth many years ago [7 p. 24]. Studies in this laboratory indicated that several antibiotics, particularly those in the polymyxin and aminoglycoside families, are inhibited by constituents of bacteriologic agar and various culture media [8]. The phenomenon appears to be related to the charge produced by free amino groups in the drugs and sulfate groups in the medium, since it can be overcome readily by addi-