Isolation and characterization of plasma membranes from guinea pig ileum
Conference
·
· Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5220117
A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca/sup 2 +/. The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na/sup +/-K/sup +/-ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca/sup 2 +/ in the presence of ATP. The addition of Ca/sup 2 +/ ionophore A23187 to vesicles loaded with Ca/sup 2 +/ in the presence of ATP removed Ca/sup 2 +/ completely from the vesicles in one minute. The Km values for the Ca/sup 2 +/ -dependent phosphorylated intermediates of Ca/sup 2 +/ -Mg/sup 2 +/ -ATPase and Ca/sup 2 +/ uptake were approximately 0.8 ..mu..M indicating that the phosphorylated intermediates represent phosphorylation of Ca/sup 2 +/ pump ATPase. The /sup 3/H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport.
- Research Organization:
- Univ. of Iowa, Iowa City
- OSTI ID:
- 5220117
- Report Number(s):
- CONF-8604222-
- Conference Information:
- Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:3
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AFFINITY
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
ATP
ATP-ASE
BODY
CALCIUM COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRIFUGATION
CHEMICAL COMPOSITION
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
GASTROINTESTINAL TRACT
GUINEA PIGS
HYDROLASES
INTESTINES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE TRANSPORT
MEMBRANES
MICROSOMES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
RODENTS
SEPARATION PROCESSES
SMALL INTESTINE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AFFINITY
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
ATP
ATP-ASE
BODY
CALCIUM COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRIFUGATION
CHEMICAL COMPOSITION
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
GASTROINTESTINAL TRACT
GUINEA PIGS
HYDROLASES
INTESTINES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE TRANSPORT
MEMBRANES
MICROSOMES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
RODENTS
SEPARATION PROCESSES
SMALL INTESTINE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES