A high-performance liquid chromatography assay of brain adenylate cyclase using [3H] ATP as substrate

A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [3H]ATP as substrate, and the principal compound contaminating the [3...

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Veröffentlicht in:	Neurochem. Res.; (United States) 1986-02, Vol.11 (2), p.161-171
1. Verfasser:	CHILDERS, S. R
Format:	Artikel
Sprache:	eng
Schlagworte:	550201 - Biochemistry- Tracer Techniques ADENOSINE Adenosine - analysis Adenosine Deaminase Adenosine Triphosphate Adenylyl Cyclases - analysis AMIDINASES AMP ANIMALS ATP BASIC BIOLOGICAL SCIENCES Biochemistry and metabolism Biological and medical sciences BODY BRAIN Brain - cytology Brain - enzymology Cell Membrane - cytology Cell Membrane - enzymology CENTRAL NERVOUS SYSTEM CHROMATOGRAPHY Chromatography, High Pressure Liquid - methods Corpus Striatum - cytology Corpus Striatum - enzymology CYCLASES Cyclic AMP - analysis ENZYMES Fundamental and applied biological sciences. Psychology HYDROLASES ISOTOPE APPLICATIONS LABELLED COMPOUNDS LIQUID COLUMN CHROMATOGRAPHY LYASES Male MAMMALS NERVOUS SYSTEM NON-PEPTIDE C-N HYDROLASES NUCLEOSIDES NUCLEOTIDES ORGANIC COMPOUNDS ORGANS Osmolar Concentration RATS Rats, Inbred Strains RIBOSIDES RODENTS SEPARATION PROCESSES Substrate Specificity SUBSTRATES TRACER TECHNIQUES Tritium TRITIUM COMPOUNDS VERTEBRATES Vertebrates: nervous system and sense organs
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Beschreibung
Zusammenfassung:	A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [3H]ATP as substrate, and the principal compound contaminating the [3H]cyclic AMP peak, adenosine, is removed by incubation of assay tubes with small amounts of adenosine deaminase. The HPLC elution utilizes high resolution (3 microns) short (10 cm) C-18 columns for increased resolution and decreased flow rates. Since cyclic AMP elutes at 4 min following injection, this procedure can easily process large numbers of samples per day when combined with automated techniques of sample injection and collection.
ISSN:	0364-3190 1573-6903
DOI:	10.1007/BF00967965