The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta

Summary The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na⁺, K⁺-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg²⁺-ATPase).Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor.It is concluded that the strontium capture technique gave a reliable localization for Na⁺, K⁺-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

The distribution of free amino acids in subcellular fractions of guinea-pig brain

1. The free amino acids of homogenates of guinea-pig brain in 0.32m-sucrose and of subcellular fractions derived therefrom have been estimated by the method of Moore & Stein. 2. Seven amino acids together accounted for over 80% of the free amino compounds; these are, in decreasing order of abundance: glutamate, aspartate, gamma-aminobutyrate, glycine, serine, alanine and threonine. In addition, there are appreciable quantities of amide (presumably glutamine). 3. Control experiments showed that the pattern of free amino acid occurrence in sucrose homogenates was similar to that of brains of animals killed by freezing in liquid nitrogen and extracted immediately without thawing. 4. The subcellular distribution of the amino acids resembled that of soluble cytoplasmic markers; there was no specific localization in a fraction rich in isolated presynaptic nerve terminals of amino acids capable of exciting or depressing central neurones. 5. The significance of the results is discussed in relation to the possible role of centrally active amino acids as transmitters.     

Studies on intestinal adenosine triphosphatases. II. Stabilitiies in different rat subcellular fractions.

Subcellular fraction (brush border, mitochondria, microsomes and plasma membranes) are isolated from the rat intestinal epithelial cells. A comparison was made between the effect of cold storage, freeze-thawing, heating and of some chemicals (DMSO, DTT, glycerol, sucrose) on the stability of Mg2+ and (Na+-K+) dependent ATPases in these fractions in order to determine possible difference linked to the localization in the enterocyte. Enzymatic activities were found more stable at -20 degrees C than at +4 degrees C. Microsomal (Na+-K+)-ATPase increased in activity until the 8th day, then declined. Brush border (Na+-K+)-ATPase was the least resistant of all fractions. For Mg2+-ATPase, that from mitochondria was that had lost much more activity (84%) in 15 days at +4 degrees C. With freeze-thawing there was a comparable decrease in all activities (20-35%). by heating between 35 and 60 degrees C, Mg2+-ATPase was shown to be more heat resistant than (Na+-K+)-ATPase. The addition of some stabilizing chemicals (DMSO, glycerol, sucrose) improved the heat stability of the two enzymes: better results were obtained with glycerol for Mg2+-ATPase and sucrose for (Na+-K+)-ATPase. These differences might be due to the compositon in membraine lipids or to the nature of the enzymes studied.     

Localization of recently characterized membrane transport adenosine triphosphatases

Summary Na⁺, K⁺-ATPase is the best-known member of a family of membrane transport ATPases sharing a number of structural features. Methods recently developed for Na⁺, K⁺-ATPase localization may, therefore, be modifiable for use in studies of the distributions of related enzymes. Our experiences in the use of monophosphate and triphosphate substrate methods for localization of the gastric proton pump ATPase (H⁺, K⁺-ATPase) are described and the prospects for localizing other related enzymes by similar techniques are discussed.     

The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain

1. The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain. 2. Glutamate decarboxylase is localized in the synaptosome fraction. The mean density of the particles containing the enzyme is slightly greater than those derived from cholinergic neurones, though overlap is substantial. 3. The enzyme is readily released from synaptosomes by hypo-osmotic treatment, but in the presence of Ca(2+), Na(+) and K(+) it sediments with particulate material. 4. The release and binding of the enzyme to membrane fractions by Ca(2+) were investigated. 5. Aspartate transaminase is present in brain as two isoenzymes with different kinetic properties. One isoenzyme is associated with the cytoplasm and the other with mitochondria.     

The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

Schistosoma mansoni: fine structural localization of tegumental adenosine triphosphatases

The distribution of Ca2+-dependent adenosine triphosphatase (EC 3.6.1.3.) and nonspecific (Na-K-Mg) adenosine triphosphatase activity in the tegument and subtegumental tissues of Schistosoma mansoni from both mixed and single sex infections was investigated cytochemically. Differences in the distribution of tegumental Ca-adenosine triphosphatase activity in 60- to 70-day-old female worms were found which could be related to the degree of sexual development in the two types of females, with little or no tegumental activity being found in 70-day-old females from single sex infections. In contrast, 28-day-old females from single sex infections showed low levels of tegumental Ca-adenosine triphosphatase activity, suggesting that the lack of tegumental activity in 70-day-old single sex females may be due to a loss or suppression of activity as a consequence of the failure of females in single sex infections to pair and develop to full sexual maturity. No differences in the distribution of nonspecific (Na-K-Mg) adenosine triphosphatase activity between females from mixed and single sex infections were found. The sexual status or age of male worms appeared to have little or no effect on the distribution of tegumental adenosine triphosphatases.     

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5-nucleotidase (5N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.     

Lysophospholipase activity in rat brain subcellular fractions

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [³²P]glycerophosphocholine from [³²P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg²⁺, Cu²⁺, and Zn²⁺ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25–200 M), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 g/mg protein and inhibition at higher concentrations. Addition of cholesterol (50–250 g/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because, of their detergent properties.     

The subcellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine

1. Two different subcellular fractionation techniques were applied to guinea-pig intestinal mucosa and the composition of the brush borders prepared by the two methods were compared. 2. By using a kinetic assay system the subcellular distribution of activity against ten dipeptides and five tripeptides was studied. 3. Only small amounts (5–10%) of activity against dipeptides were found in the brush-border region, the enzymes being concentrated in the cytosol. 4. Significant amounts (10–60%) of activity against tripeptides were found in the brush border with the remainder largely present in the soluble fraction. 5. The relevance of these studies to the localization in vivo and the possible role of peptidases in protein digestion is discussed.