ATPase, marker

Maier WE, Costa LG. 1990. Sodium, potassium-ATPase in rat brain and erythrocytes as a possible target and marker, respectively, for neurotoxicity studies with chlordecone, organotins and mercury compounds. Toxicol Lett 51 175-188. 

Certain enzymes shown to be present in myelin could be involved in ion transport. Carbonic anhydrase has generally been considered a soluble enzyme and a glial marker but myelin accounts for a large part of the membrane-bound form in brain. This enzyme may play a role in removal of carbonic acid from metabolically active axons. The enzymes 5 -nucleotidase and Na+, K+-ATPase have long been considered specific markers for plasma membranes and are found in myelin at low levels. The 5 -nucleotidase activity may be related to a transport mechanism for adenosine, and Na+, K+-ATPase could well be involved in transport of monovalent cations. The presence of these enzymes suggests that myelin may have an active role in ion transport in and out of the axon. In connection with this hypothesis, it is of interest that the PLP gene family may have evolved from a pore-forming polypeptide [9],... 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

Krenacs, T., Stiller, D., Krenacs, L., Bahn, H., Molnr, F., and Dux, L. (1990) Sarcoplasmic reticulum (SR) Ca +-ATPase as a marker of muscle-cell differentiation immunohistochemical investigations of rhabdomyosarcomas and enhancement of immunostaining after sodium methoxide treatment. Acta Histochem. 88, 159-166. 

To characterize the preparation, determine marker enzymes, e.g., p-nitrophenyl phosphatase (PNPase), ouabain-sensitive Na,K-dependent ATPase, or dihydropyridine receptor complex (L-type voltage dependent calcium channel). 

Determination of a Marker Enzyme Ouabain-Sensitive Na,K-ATPase... 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

Ca2 +-ATPase has been used as an enzymic marker for the brush border of H. diminuta, although its precise function is open to question. Two forms of this enzyme occur within the tegument of the parasite. One appears to be calmodulin dependent and is active at a higher pH than the other (Table 6.2). Calmodulin appears to be a ubiquitous Ca2 + receptor which plays a part in most of the Ca2+-regulated processes that have been studied in eukaryotic cells. It is a highly conserved protein, so it is not surprising that... 

Acidifies the cytoplasm and freezes clathrin networks Blocks budding of clathrin-coated vesicles Inhibits macropinocytosis Inhibits v-ATPases raises endosomal pH Fluorescent marker of the golgi complex Inhibitor of protein transport in the golgi complex Weak base raises endosomal pH disrupts endosomes Inhibits clathrin lattice formation Disrupts clathrin lattices... 

Membranes of extremely halophilic bacteria oxidize NADH in the presence of a variety of redox dyes, including menadione [103] and DCIP [100]. The enzyme, which is located on the inner aspect of the cytoplasmic membrane [104], has been used as a marker of vesicle orientation [20]. However, a second marker should be used in conjunction with menadione reductase as there are indications that the location of the dehydrogenase may be randomized during membrane preparation [105]. A similar situation occurs with respect to the ATPase activities in Micrococcus lysodeikticus [106] and E. coli [75,107] as well as the NADH and succinate dehydrogenases from E. co/f [75,108]. The orientation of the NADH dehydrogenase in the case of H. saccharovorum does not correspond... 

Proximal tubule cells in culture should have retained functional attributes such as (1) polar architecture and junctional assembly of epithelia and correct membrane distribution of enzymes and transport systems (2) vectorial transport of solutes and water, manifested by the formation of domes when cultured on solid supports [81] and the generation of transepithelial electrophysiological properties [82, 83] due to the expression of proximal tubule specific claudins 2- and 10 [84, 85] (3) cellular uptake of xenobiotics from either the apical or basolateral side, as observed in vivo and (4) expression of nephron segment-specific characteristics, i.e., distinct expression of differentiation markers, metabolic and transport properties, and hormone responsiveness. Such markers include the expression of the brush border enzymes alkaline phosphatase, leucine aminopeptidase, and y-glutamyl transferase [4, 86], In addition, proximal tubule cells should possess Na+,K+-ATPase activities, Na+-dependent glucose, and p-aminohippurate transport. Proximal tubule cells increase cAMP levels in response to parathyroid... 

Andersson G, Lindunger A, Ek-Rylander B (1989) Isolation and characterization of skeletal acid ATPase-a new osteoclast marker Connect Tissue Res 20 151-158... 

B9. Berg, P. A., Klein, R., Lindenborn-Fotinos, J., and Kloppel, W., ATPase-associated antigen (M2) Marker antigen for serological diagnosis of primary biliary cirrhosis. Lancet 2, 1423— 1425 (1982). 

The apical plasma membrane of epithelial cells of small intestinal and renal proximal tubules is characterised by the presence of many microvilli (brush border). These membranes can be isolated relatively easily by centrifugation and free flow electrophoresis techniques. Kinne-Saffran and Kinne [15] found that after free-flow electrophoresis of a rat kidney-cortex membrane preparation, the anion-sensitive ATPase co-migrated with the alkaline phosphatase activity but was separated from the (Na + K )-ATPase activity, which is assumed to be a marker of basolateral plasma membranes. This suggests that the brush-border membrane of the proximal tubule contains an anion-sensitive ATPase. The same conclusion was reached by Liang and Sacktor [17] for a brush-border preparation from rabbit kidney. 

Upon purification, the K -stimulated phosphatase activity is always copurified with the (K )-ATPase activity [63-65]. Mitochondrial markers, such as cytochrome c oxidase, succinate dehydrogenase, monoamino-oxidase, and the ribo-somal marker RNA are largely removed by the purification procedure. The same is true for the anion-sensitive ATPase and 5 nucleotidase activities, but some (Na — K )-ATPase activity is still present in highly purified (K" -I-H )-ATPase preparations. Purification is also characterised by a lowering of the K -insensitive Mg ATPase activity, but even in the purest preparations some Mg -ATPase activity (4% of (K -I- H )-ATPase activity) is still present. This may represent an impurity or an inherent property of the enzyme. 

A two- to three-fold increase in Na -K -ATPase activity, a nonspecific marker a three-fold reduction in a negative marker, alkaline phosphatase identified with proximal tubular cells and immuno-fluorescent localization of Tamm-Horsfall protein, a specific marker for mTALH cells. 

---