Receptor-ligand interactions studied with homogeneous fluorescence-based assays suitable for miniaturized screening.

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness.

Two-dimensional fluorescence intensity distribution analysis: theory and applications.

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses.

Production of the immunoglobulin variable domain REIv via a fusion protein synthesized and secreted by Staphylococcus carnosus.

REIv--the variable domain of an immunoglobulin x light chain--was produced by heterologous gene expression in a Gram-positive bacterium, purified to homogeneity and characterized. A host/vector combination based on secretion of Staphylococcus hyicus lipase by Staphylococcus carnosus was exploited. A gene encoding a fusion protein, composed of an aminoterminal portion of the pre-pro-peptide of S.

The effect of zinc in vivo and in vitro on the activities of angiotensin converting enzyme and kininase-I in the plasma of rats.

Two groups of rats were pair fed, for 18 days, diets containing either 2.6 (Zn deficient) or 100 mg Zn/kg (control diet). Plasma was assayed spectrophotometrically for the activity of kininase-I and angiotensin converting enzyme (ACE) in the presence of varying concentrations of added Zn2+.

Evidence for a potent angiotensin I degrading enzyme different from angiotensin I converting enzyme in rat vascular tissues.

There are indications for the existence of an intrinsic renin angiotensin system in vascular walls, which is assumed to participate in blood pressure regulation and in pathogenesis of arterial hypertension. It was evaluated if and to what extent the decapeptide angiotensin (A) I, one of the natural substrates of A I converting enzyme (ACE), is degraded by other peptidases than ACE in rat vascular tissues. A I and A II degradation was studied in arterial and venous vascular wall extracts.

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Investigations were performed on components of the renin-angiotensin system (RAS) in homogenate extracts of vascular tissue and aortic smooth muscle cells cultivated in vitro. Determinations of isoelectric points and pH optima indicated the existence in aortic homogenate extracts of two local angiotensin I (AI)-forming enzymes (AIFE) that were different from those of plasma, renal cortex, veins, and aortic smooth muscle cells. The pH optima for AI-converting enzyme (ACE) from vascular tissues, aortic smooth muscle cells, and plasma were in the same range (pH 8.

Angiotensin I converting enzyme in the ejaculate of fertile and infertile men.

ACE has been measured in the seminal plasma of fertile and infertile men according to the method of Cushman and Cheung. High enzyme activity was found in fertile subjects which did not significantly differ from those with oligozoospermia and those with the Sertoli cell-only syndrome. Enzyme activity was significantly lower after vasectomy.