Large scale partial purification and molecular and kinetic properties of the (Na + K)-activated adenosine triphosphatase from Artemia salina nauplii
The sodium-potassium coupled transport enzyme (NaK-ATPase) has been partially purified from the nauplius larva of the brine shrimp, Artemia salina, by treatment of isolated membranes with deoxycholate, followed by a new freeze-thaw procedure. Purification is up to 200-fold (relative to the homogenate), with a yield of 10 mg of protein from 200 g, wet weight, of brine shrimp nauplii. The enzyme preparations have specific activities up to 475 ..mu..mol of Pi/h/mg of protein, with an estimated 50% purity, assuming both the large and small subunits are the protein constituents of the holoenzyme. Two proteins enrich during purification, the larger of which is identified as the catalytic subunit by its potassium-sensitive, (Na + Mg)-dependent phosphorylation from (..gamma..-/sup 32/P)ATP. The mass ratio is estimated to be greater than two, corresponding to a molar ratio of one large to one small subunit. Binding studies indicate that the preparation has an equivalent number of phosphorylation sites and ATP binding sites. The calculated molecular weight of a theoretically purified enzyme based on the binding studies is 230,000 and 220,000, respectively, for the two ligands. The binding studies and the subunit molecular weight amd mass ratio analysis suggest that the enzyme is an ..cap alpha../sub 2/..beta../sub 2/ tetramer, of which only half of the sites are apparently reactive at one time. The subunits are consistently double-banded on sodium dodecyl sulfate gels, and the potassium-sensitive phosphorylation reaction appears in both bands of the large subunit. The evidence suggests there may be two NaK-ATPases in the brine shrimp.
- Research Organization:
- Oregon State Univ., Corvallis
- OSTI ID:
- 6328890
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 253:13
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
BIOCHEMICAL REACTION KINETICS
PHOSPHORUS 32
TRACER TECHNIQUES
ENZYME ACTIVITY
MOLECULAR WEIGHT
POTASSIUM
PURIFICATION
SHRIMP
SODIUM
STRUCTURAL CHEMICAL ANALYSIS
ACID ANHYDRASES
ALKALI METALS
ANIMALS
AQUATIC ORGANISMS
ARTHROPODS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CRUSTACEANS
DAYS LIVING RADIOISOTOPES
DECAPODS
ELEMENTS
ENZYMES
HYDROLASES
INVERTEBRATES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
METALS
NUCLEI
ODD-ODD NUCLEI
PHOSPHOHYDROLASES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
550201* - Biochemistry- Tracer Techniques