Surface-bound biomembranes incorporating receptors: electrochemical and structural characterization.

A generic method is described for forming surface-bound structures that incorporate protein receptors in a membrane-like environment. Silane reagents (octadecyltrichlorosilane and dimethyloctadecylchlorosilane) were used to produce primed substrates bearing full and partial monolayers, respectively. Biomembranes were formed by dialysis of detergent-solubilized membranes in the presence of two different alkylsilanized substrates: Si/SiO2 electrodes and glass microspheres. Electrochemical analysis of the capacitance was used to determine apparent thickness and degree of surface coverage at each stage in the deposition process. Elemental analysis on glass beads gave the hydrocarbon incorporation. Glass bead substrates were also examined by Fourier transform infrared spectroscopy to evaluate the alkylsilanized substrate before and after dialysis. Both vertebrate rhodopsin and the nicotinic acetylcholine receptor could be incorporated into structures with composition and dimensions similar to natural bilayer membranes. The techniques reported here are applicable for coupling membrane receptors to a variety of transducing substrates used in biosensors.     

Electrophoretic separation and quantitation of creatine kinase isozymes

A method is described for separating and quantitating the isozymes of creatine kinase in various tissue extracts. We have tested the sensitivity and precision of the method over a wide range of mixtures of isozymes. The quantitation is linear in the range of 0–5 mIU of CK analyzed, and the lower limit of quantitation of an isozyme in a mixture is 10 mIU/ml. For certain tissues the presence of dithiothreitol is necessary to prevent inactivation of the brain-type isozyme.     

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

On the origins of esterases

Comparisons among the primary sequences of five cloned eukaryotic esterases reveal two distinct lineages, neither bearing any significant overall sequence similarity to the functionally related serine protease multigene family. We have not eliminated the possibility that the esterases may have residual conformational similarities to the serine proteases. However, our profile analysis and analyses of the predicted conformations of the esterases reveal little similarity to the serine proteases. Four of the esterase proteins share 27%-53% overall sequence similarity and evidence of a catalytic mechanism involving the same Arg- Asp-Ser or His-Asp-Ser charge relay. We propose that these four esterases, three of them cholinesterases, form part of a multigene family essentially separate from the serine proteases.      

The growth-supporting activity of a retinoidal benzoic acid derivative and 4,4-difluororetinoic acid

Two synthetic retinoids were examined for their ability to support growth in male vitamin A-deficient rats. One of the compounds, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 -propenyl]-benzoic acid (TTNPB), was found to be highly effective; it was 35-fold more active than all-trans-retinoic acid. Thus, the in vivo results were in agreement with the in vitro activity of this compound published by previous investigators, and support the view that this compound may be useful in determining the molecular mechanism of action of the retinoids. Another analog, 4,4-difluororetinoic acid, was only 12% as effective as retinoic acid. However, the possible instability of this compound and the electronegativity of the fluoro groups prohibited conclusions concerning the biological function of metabolic modification on the 4 position of retinoic acid.