Kenneth R. Case

Diazepam receptor characterization: Specific binding of a benzodiazepine to macromolecules in various areas of rat brain

Diazepam is an effective anti-anxiety drug or mild tranquilizer [l] which is also used as a muscle relaxant [2] , a presurgery medication [3], an anti-epileptic agent and in alcohol withdrawal. It is a widely prescribed drug [4]. The toxicity of diazepam is remarkably low, as is its physical dependence [S] . Despite its widespread use, very little is known about the molecular basis of its action. Recent investigations, concerning macromolecules that bind hormones or drugs, have been carried out in an attempt to correlate such binding with pharmacological actions. If a binding macromolecule is found for a drug, it is usually designated as a receptor, and it is assumed that an endogenous or naturally occurring ligand exists for the receptor. In the present study we found evidence for a highly specific, saturable binding of [3H]diazepam to a purified synaptosomal fraction from rat forebrain and present some of the characteristics of this binding. (A note of caution should be interjected since there is a great deal of difference between a binding entity, a receptor and a receptor site. In the past, glass, plastics, and other inert materials have been shown to be effective ‘binders’ of ligands.) Such data should lead to a better understanding of the molecular action of diazepam. While this work was nearing completion, Squires and Braestrup [6] reported on benzodiazepine receptors in rat brain. In our report, using a higher specific activity of [3H]diazepam and a more purified binding fraction, we give amplified details of the diazepambinding site. 2. Materials and methods

Diazepam receptor: Specific binding of [3H]diazepam and [3H]flunitrazepam to rat brain subfractions

Evidence is beginning to accumulate that specific molecules bind the benzodiazepines in a receptorrelated manner [l-4]. However, to date, no studies have demonstrated to which cellular structures the diazepam was bound. Morphologic confirmation is essential when one is dealing with the central nervous system, since ‘standard’ fractionation procedures for one area in the brain give varying results in other regions of the brain [5]. We report here that the fraction of rat forebrain containing synaptic junctions binds specifically [3H]diazepam; furthermore, we show that [3H]flunitrazepam binds to the benzodiazepine ‘receptors’ with more than IO-times greater affinity in the described system than [3H]diazepam and hence may be a useful tool for further investigation.

Mitochondrial autonomy: incorporation of monosaccharides into endogenous glycolipid acceptors in isolated rat liver mitochondria.

Abstract Isolated, sterile rat liver mitochondria homogenized in Triton X-100 incorporated glucose-14C and mannose-14C into endogenous lipid acceptors forming glycolipids. In addition, choline-14C was incorporated into phosphatidyl choline in the system. The glycolipid incorporations were highly specific and showed an absolute requirement for Mg++. One of the products formed with each monosaccharide was identified as a diglyceride glycolipid. Fractionation of the mitochondria into inner and outer membrane fractions indicated that the choline, glucose and mannose systems were associated with the outer membranes of the mitochondria.

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Abstract Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.

Cell surface changes caused by growth of B16 melanoma cells in bromodeoxyuridine

Most normal cells have cell surface properties different from their transformed or oncogenic counterparts (reviewed in [l]). While tumor cells will grow without being attached to a substratum, most untransformed cells require a surface to which they can &here in order to grow [2,3]. Variations in cell adhesion mediated by changes in the cell surface could play a determinant role in the regulation of cell growth and development as well as in the mechanism of oncogenesis. Bromodeoxyuridine (BrdUrd), a thymidine analogue, exerts a selective and reversible effect on specific cellular differentiative functions and oncogenic potential (reviewed in [4] and [5] ). Although both DNA-linked and non-DNA-mediated effects of BrdUrd have been described [4], the mechanism of action of this drug is unknown. While much information has been gathered on the effects of BrdUrd on DNA structure, much less attention has been paid to BrdUrd’s effects on the plasma membrane. In order to evaluate quantitatively the effect of BrdUrd on the plasma membrane, adhesivity of mouse melanoma cells was measured as well as their electrophoretic mobility and aqueous polymer partitioning behavior. The results show that BrdUrd treatment greatly increases the adhesivity of these cells without changing their electrophoretic mobility. Treatment with BrdUrd does, however, change the partition ratio of these cells in aqueous two-phase dextran-polyethylene glycol suggesting that basic alterations of the cell surface have occurred.

Surface electrokinetic properties of two strains of Staphylococcus aureus during their cell growth cycle

Abstract Two strains of Staphylococcus aureus were investigated: S. aureus H, a normal wild-type strain, and 52A5, a mutant strain whose cell wall contains no teichoic acid but is made up entirely of mucopeptide. S. aureus H cells in the lag or stationary phase of growth had an electrophoretic mobility of −1.10 μm/s/V/cm while those in the logarithmic phase had a mobility of −0.80 μm/s/V/cm in saline at pH 7.2, 0.6 mM NaHCO 3 , 25°C (I = 0.145 g-ions/l). S. aureus 52A5 cells in the same solution had a mobility of −0.87 μm/s/V/cm in lag and stationary growth phases but a mobility of −1.30 μm/s/V/cm in the logarithmic growth phase. The S. aureus H cell surfaces at lag phase had pKs of 3.2 and 9.5; at logarithmic phase, 4.2 and 9.0; and at stationary phase, 3.0 and 9.5. The 52A5 cell surfaces at lag phase had pKs of 2.3 and 10.3; at logarithmic phase, 1.7 and 8.5; at stationary phase, 2.6 and 10.2.