METABOLISM OF EXTERNAL ADENINE NUCLEOTIDES BY HUMAN RED BLOOD CELLS
METABOLISM OF EXTERNAL ADENINE NUCLEOTIDES BY HUMAN RED BLOOD CELLS
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DOI：
10.1152/ajplegacy.1970.218.6.1568
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发表时间：
1970-01-01
影响因子：
--
通讯作者：
PARKER, JC
中科院分区：
文献类型：
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作者：
PARKER, JC
METHODSThe following labeled nucleotides were obtained from Amersham-Searle Corp., Des Plaines, 111.: AM32P, 350 mc/mmole; AMP-8-14C, 53 mc/mmole; AMP-U-14C, 135 mc/mmole. ATP-8-14C, 15.6 mc/mmole, and ADP-8-14C, 11 mc/mmole, were purchased from Calbiochem, Los Angeles, Calif. The purity of the radioactive compounds was assessed by diethylaminoethyl(DEAE) cellulose paper chromatography (see below). More than 97% of all the isotopes migrated with authentic compound except for ATP-8-14C, which contained a 15% admixture of ADP. Nonradioactive nucleotides, nucleosides, and purines were obtained from Sigma Chemical Company, St. Louis, MO. Adenosine deaminase was purchased from Boehringer Mannheim Corp., New York.The solution which was used as a wash and as a basic incubation medium for most of the experiments with intact cells contained, in millimoles per liter, NaCl, 145; KCl, 5; MgSO4, 1; glycylglycine, 20; and glucose, 10; at pH 7.5. For studies of pH dependence, the medium was altered by substituting for glycylglycine a mixture of tris (hydroxymethyl) methylaminopropane sulfonic acid (10 mM) and 2-(N-morpholino) ethanesulfonic acid (10 mM)(12). These buffers were obtained from Calbiochem. Blood from hematologically normal human adults was drawn into syringes rinsed with heparin (5,000 U/ml) and immediately injected into a glass column packed with adsorbent cotton (5). The column was sealed and incubated for 30 min in a 37 C oven. The red cells were eluted from the column with wash solution at 37 C and washed 4 times by slow centrifugation at 10 C, the top layer of cells being discarded. Following this procedure, less than 100 leukocytes and no platelets could be detected per cubic millimeter of packed red blood cells by visual and electronic methods.
