Albert Missbichler

Light-independent NADPH-protochlorophyllide oxidoreductase activity in purified plasma membrane from the cyanobacterium Anacystisnidulans

A light plasma membrane fraction corresponding to a buoyant density of 1.087 +/- 0.005 g/cm3 and devoid of chlorophyll was prepared and purified from Anacystis nidulans according to a recently published procedure (G.A.Peschek, V.Molitor, M.Trnka, M. Wastyn and W. Erber (1988) Methods Enzymol. 167, 437-449). Besides major amounts of carotenoids the plasma membranes contained a small but significant pool of chlorophyllide a and protochlorophyllide a as verified by room temperature and 77K spectrofluorimetry and analytical separation and identification by high performance liquid chromatography using authentic standards. Incubation of the plasma membranes in strict darkness in the presence of NADPH was accompanied by the gradual and stoichiometric replacement of protochlorophyllide by chlorophyllide, NADP+ effecting the reverse transition. The reaction was completely insensitive to illumination (5-20 w/m2 tungsten light) but abolished after heating of the membranes (90 degrees C, 5 min) or in the presence of 10 mM EGTA, and was specifically stimulated by calcium ions. Our results indicate the occurrence of light-independent NADPH:protochlorophyllide oxidoreductase activity in the plasma membrane of Anacystis nidulans.

Determination of α-human atrial natriuretic peptide (α-hANP) in urine using combination of HPLC with RIA strongly indicates non-immunoreactive metabolites

Abstract Employing HPLC coupled with RIA, it was shown that α-human atrial natriuretic peptide is excreted in urine. Freshly collected urine had to be acidified to obtain reproducible results. When prepurified urine was subjected to HPLC (ion exchange and reversed phase) the subsequent quantification of α-hANP immunoreactive material in the eluate showed 10- to 30-fold greater amounts of α-hANP after treatment with HPLC; substances with the same elution parameters as synthetic α-hANP were detected, but they gave no response in the RIA.

Immobilization of α-human atrial natriuretic peptide on insoluble supports and affinity purification of specific antibodies from a polyclonal goat anti-α-human atrial natriuretic peptide serum

Abstract α-Human atrial natriuretic peptide (α-hANP) was covalently coupled via single attachment onto two different insoluble matrices. Controlled-pore glass—α-hANP matrices were well suited for the purification of monospecific antibodies, whereas Enzacryl AA—α-hANP did not withstand the inevitable chemical and physical stresses during affinity purification.

Construction and use of two α-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype α-hANP biosensor

Abstract Two α-human atrial natriuretic peptide (α-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of α-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate α-hANP-specific antibodies from a goat anti-α-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an α-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological α-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This α-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.