scholarly journals The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish

1975 ◽  
Vol 151 (1) ◽  
pp. 61-66 ◽  
Author(s):  
P Ottolenghi
Keyword(s):  
Adenosine Triphosphatase ◽  
Gel Filtration ◽  
Salt Gland ◽  
Enzymic Activity ◽  
Potassium Ion ◽  
Soluble Form ◽  
Spiny Dogfish ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Tissue Fraction

A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.

scholarly journals The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

1975 ◽  
Vol 23 (8) ◽  
pp. 571-574 ◽  
Author(s):  
J A Firth ◽  
B Y Marland
Keyword(s):  
Alkaline Phosphatase ◽  
Adenosine Triphosphatase ◽  
Renal Cortex ◽  
Sodium Potassium ◽  
Cytochemical Demonstration ◽  
Nitrophenyl Phosphate ◽  
Atpase Cytochemistry ◽  
Strontium Ions ◽  
Dependent Transport ◽  
Hydrolysis Of

The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

scholarly journals TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY I. BIOCHEMICAL CHARACTERIZATION OF A CYTOCHEMICAL MEDIUM FOR THE ULTRASTRUCTURAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE AVIAN SALT GLAND

1972 ◽  
Vol 20 (1) ◽  
pp. 13-22 ◽  
Author(s):  
STEPHEN A. ERNST
Keyword(s):  
Enzymatic Activity ◽  
Adenosine Triphosphatase ◽  
Biochemical Characterization ◽  
Metal Salt ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Defined Medium ◽  
Fixed Tissue ◽  
Fold Reduction ◽  
Nitrophenyl Phosphate

The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.

TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY II. CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE SECRETORY EPITHELIUM OF THE AVIAN SALT GLAND

1972 ◽  
Vol 20 (1) ◽  
pp. 23-38 ◽  
Author(s):  
STEPHEN A. ERNST
Keyword(s):  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Defined Medium ◽  
Secretory Cells ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Nitrophenyl Phosphate

A cytochemical procedure is described for the ultrastructural localization of K-dependent, ouabain-sensitive nitrophenyl phosphatase activity in avian salt gland. Cryostat sections (50 µ) of paraformaldehyde-fixed tissue were incubated in a kinetically defined medium containing: 5 mM p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM KCl, 100 mM Tris-HCl buffer (pH 8.5 or 9.0) and 20 mM SrCl2 to precipitate hydrolyzed phosphate. After incubation at room temperature, the sections were treated with Pb(NO3)2 to convert SrPi to PbPi precipitates for visualization in the electron microscope. Reaction product was localized on the cytoplasmic side of the secretory cell lateral and basal plasma membranes. Little, if any, reaction product was associated with the apical surfaces of the secretory cells or with endothelial surfaces of capillaries. Appropriate control experiments indicated that deposition of reaction product was dependent on Mg and K and was sensitive to ouabain. Furthermore, nonenzymatic hydrolysis of nitrophenyl phosphate did not occur in the medium, and deposition of artifactually produced precipitates did not resemble deposition of enzymatically produced precipitates. The relationship of this localization to transport adenosine triphosphatase cytochemistry is discussed, and the physiologic implications of the localization for tracing the route of active Na transport in the salt gland are considered.

scholarly journals Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis.

1975 ◽  
Vol 23 (11) ◽  
pp. 828-839 ◽  
Author(s):  
R Beeuwkes ◽  
S Rosen
Keyword(s):  
Atpase Activity ◽  
Electron Probe ◽  
Sequential Analysis ◽  
Relative Sensitivity ◽  
Initial Reaction ◽  
Initial Product ◽  
Sodium Potassium ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Convoluted Tubules

The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments.

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