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.

Ultrastructural Localization of NA+-K+ -Atpase in Rabbit Cornea Using 5-Nitroindoxyl Phosphate as Substrate

1977 ◽  
Vol 35 ◽  
pp. 444-445
Author(s):  
K. C. Tsou ◽  
Mahin Khatami
Keyword(s):  
Endothelial Cells ◽  
Comparative Study ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Endothelial Damage ◽  
Previous Method ◽  
New Method ◽  
Rabbit Cornea ◽  
Fixed Tissue ◽  
Nitrophenyl Phosphate

Recently, Leunberger and Novikoff (1) have shown that the endothelial cells are the primary sites of Na+-K+-ATPase, a key mechanism in the control of hydration of cornea. Since this enzyme would be a useful marker for our study of endothelial damage by CO2 laser (2), we have undertaken a comparative study for the localization of this enzyme at ultrastructural level with different methods. In the course of this work we developed a new method with the use of 5-nitroindoxyl phosphate as a synthetic substrate. This new substrate, like p-nitrophenyl phosphate (3), meets the requirements for the demonstration of the K+ activated step of dephosphorylation of ATPase. In addition, it can be used with glutaraldehyde fixed tissue, thereby offering an important advantage over the previous method (3).Rabbit corneas were freshly excised under pentobarbitol anesthesia.

scholarly journals PRESERVATION OF Na-K-ACTIVATED AND Mg-ACTIVATED ADENOSINE TRIPHOSPHATASE ACTIVITIES OF AVIAN SALT GLAND AND TELEOST GILL WITH FORMALDEHYDE AS FIXATIVE

1970 ◽  
Vol 18 (4) ◽  
pp. 251-263 ◽  
Author(s):  
STEPHEN A. ERNST ◽  
CHARLES W. PHILPOTT
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Electron Microscopic Examination ◽  
Salt Gland ◽  
Chloride Cells ◽  
Secretory Cells ◽  
Salt Glands ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Gill Filaments

The effect of glutaraldehyde and formaldehyde fixation on the level of biochemically demonstrable Na-K-adenosine triphosphatase (Na-K-ATPase) and Mg-ATPase of avian salt glands and teleost gill filaments was studied. Sections, 100-200 µ, prepared with the Smith-Farquhar tissue chopper, were fixed for varying periods, homogenized and assayed for ATPase activity. Fixation of salt gland tissue with 0.5% glutaraldehyde for 40-60 min completely inhibited the Na-K-ATPase activity and reduced the level of Mg-ATPase by 85%. In contrast, fixation with 2 or 3% formaldehyde, prepared from paraformaldehyde, for 60-90 min resulted in a loss of only 30% of the Na-K-ATPase activity and 65% of the Mg-ATPase activity. Similar results were obtained with gill filaments. In addition, Na-K-ATPase of formaldehyde-fixed tissue retained an obligatory requirement for Na+ and K+ and was fully sensitive to ouabain. Electron microscopic examination of formaldehyde-fixed tissue, sectioned with either the tissue chopper or in the cryostat and incubated in the Wachstein-Meisel medium, showed excellent morphologic preservation. Reaction product deposition (presumably due to Mg-ATPase) was associated with the extracellular side of the plasma membrane in the secretory cells of the salt gland and over the mitochondrial matrix of chloride cells present in the gill epithelium.

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 Cytochemical demonstration of sodium, potassium-adenosine triphosphatase by a hemepeptide derivative of ouabain.

1978 ◽  
Vol 26 (12) ◽  
pp. 1042-1052 ◽  
Author(s):  
J E Mazurkiewicz ◽  
F E Hossler ◽  
R J Barrnett
Keyword(s):  
Plasma Membranes ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Fixed Tissue ◽  
Sodium Potassium ◽  
Cytochemical Demonstration ◽  
Biological Affinity ◽  
Noncompetitive Inhibitor ◽  
Membrane Components ◽  
Apical Membranes

A cytochemical probe for the ultrastructural localization of the NaKATPase was devised, which utilizes the biological affinity of the noncompetitive inhibitor, ouabain (ouab) to which was coupled a hemepeptide (H11P) which possesses peroxidatic activity. The conjugate, ouab-H11P, had an apparent Ki of approximately 8 x 10(-7) M. When reacted with fixed tissue from the salt gland of osmotically stressed ducklings, the NaKATPase was localized to the basal and lateral infoldings of the plasma membranes of secretory epithelial cells. Reaction product consisted of fine textured deposits distributed in focal patches on the outer aspects of the membrane. Apical membranes were negative, as were intracellular membrane components. Preincubation of tissue with unlabeled ouabain or binding of ouab-H11P in the presence of 10 mM K+, no ATP and no Mg++, resulted in the absence or diminution of reaction product.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

scholarly journals Biochemical characterization of selected plant species from Brazilian Savannas

2004 ◽  
Vol 47 (2) ◽  
pp. 253-259 ◽  
Author(s):  
Samantha Salomão Caramori ◽  
Claudinei Sousa Lima ◽  
Kátia Flávia Fernandes
Keyword(s):  
Enzymatic Activity ◽  
Plant Species ◽  
Biochemical Characterization ◽  
Hymenaea Courbaril ◽  
Annona Crassiflora

The aim of this work was to analyze and quantify the presence of antinutritional compounds such as lectins and trypsin-like inhibitors, polyphenols and tannins, and enzymatic activity of peroxidases and proteases in the seeds of Annona crassiflora Mart. (araticum), Hymenaea courbaril L. var. courbaril (jatobá), Plathymenia reticulata Benth. (vinhático), Zanthoxylum rhoifolium Lam. (maminha de porca), Apeiba tibourbou Aubl. (pau jangada), Salacia crassiflora Mart G. Don. (bacupari), and Sclerolobium paniculatum Vog. (carvoeiro). The results suggested that these plants could be used as new source of food.

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