The effect of transforming growth factor-beta on the alkaline phosphatase activity in rabbit renal cortical tubular cell cultures

The effect of various growth regulators including epidermal growth factor and transforming growth factor-beta on the alkaline phosphatase activity of rabbit renal cortical tubular cells has been investigated in a serum-free culture. As a result, it was found that transforming growth factor-beta, known to be a growth inhibitor of renal tubular cells, increased the alkaline phosphatase activity of the tubular cells dose-dependently and that cycloheximide blocked any increase in the activity of this factor. In contrast, epidermal growth factor decreased the alkaline phosphatase activity in the tubular cells.     

Cytochemical localization of ouabain-sensitive (K+)-dependent p-nitrophenyl phosphatase (transport ATPase) in human blood platelets

The ultrastructural cytochemical localization of a potassium-dependent oubain-sensitive nitrophenyl phosphatase (transport ATPase) activity in human blood platelets is described. This potassium-dependent nitrophenyl phosphatase activity was not affected by 5 mM levamisole, indicating that the reaction product identified was not due to nonspecific alkaline phosphatase activity. The K+-dependent nitrophenyl phosphatase was strictly localized to the platelet plasma membrane, while the open canalicular system and dense tubular system were devoid of reaction product. In contrast, (Ca2+,Mg2+)-activated ATPase activity was predominantly localized in the open canalicular system and dense tubular system with very little cytochemical activity expressed at the plasma membrane. These data demonstrate a relative segregation of these enzymes into unique membrane compartments of the human platelet. Such data may be useful with regard to identification of purified membrane fractions from platelets and may be significant with regard to the understanding of the function(s) of the different membrane compartments of the human platelet.     

Stimulation of canine kidney BBMV ATPase activity by acidic pH in the presence of Zn2+: an ATPase activity distinct from transport ATPases and alkaline phosphatase that may be an ecto-ATPase

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.     

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.     

Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro

Interphase Xenopus egg extracts form extensive tubular membrane networks in vitro. These networks are identified here as endoplasmic reticulum by the presence of ER resident proteins, as shown by immunofluorescence, and by the presence of single ribosomes and polysomes, as shown by electron microscopy. The effect of phosphorylation on ER movement in interphase was tested using the phosphatase inhibitor, okadaic acid. Okadaic acid treatment resulted in an increase of up to 27-fold in the number of ER tubules moving and in the extent of ER networks formed compared to control extracts. This activation was blocked by the broad-specificity kinase inhibitor 6- dimethylaminopurine. Okadaic acid had no effect, however, on the direction of ER tubule movement, which occurred towards the minus end of microtubules, and was sensitive to low concentrations of vanadate. Inhibition of phosphatases also had no effect on the speed or duration of ER tubule extensions, and did not stimulate the activity of soluble cytoplasmic dynein. The sensitivity of ER movement to okadaic acid closely matched that of protein phosphatase 1. Although the amount of ER motility was greatly increased by inhibiting protein phosphatase 1 (PP1), the amount of cytoplasmic dynein associated with the membrane was not altered. The data support a model in which phosphorylation regulates ER movement by controlling the activity of cytoplasmic dynein bound to the ER membrane.     

Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.     

Glomerulotubular balance, dietary protein, and the renal response to glycine in diabetic rats

The glomerular filtration rate (GFR) normally increases during glycine infusion, which is a test of "renal reserve." Renal reserve is absent in diabetes mellitus. GFR increases after protein feeding because of increased tubular reabsorption, which reduces the signal for tubuloglomerular feedback (TGF). Dietary protein restriction normalizes some aspects of glomerular function in diabetes. Renal micropuncture was performed in rats 4-5 wk after diabetes was induced by streptozotocin to determine whether renal reserve is lost as a result of altered tubular function and activation of TGF, whether 10 days of dietary protein restriction could restore renal reserve, and whether this results from effects of glycine on the tubule. TGF activation was determined by locating single-nephron GFR (SNGFR) in the early distal tubule along the TGF curve. The TGF signal was determined from the ionic content of the early distal tubule. In nondiabetic rats, SNGFR in the early distal tubule increased during glycine infusion because of primary vasodilation augmented by increased tubular reabsorption, which stabilized the TGF signal. In diabetic rats, glycine reduced reabsorption, thereby activating TGF, which was largely responsible for the lack of renal reserve. In protein-restricted diabetic rats, the tubular response to glycine remained abnormal, but renal reserve was restored by a vascular mechanism. Glycine affects GFR directly and via the tubule. In diabetes, reduced tubular reabsorption dominates. In low-protein diabetes, the vascular effect is enhanced and overrides the effect of reduced tubular reabsorption.     

Ultrastructural studies on the Malpighian tubule of the pill millipede,Glomeris marginata (Villers)

Summary Ultrastructural studies on the Malpighian tubules of Glomeris marginata (Villers) reveal considerable morphological differences between the upper, fluid secreting, segment, and the lower segment which is at present of unknown function. Previous reports have shown that the upper tubule has a high permeability to compounds of high molecular weight. This may be accounted for by the fact that the epithelium shows very extensive intercellular spaces which are linked directly to junctions apparently specialised to provide a low resistance extracellular pathway between the haemocoel and the tubule lumen.Histochemical studies on the localisation of phosphatase enzymes reveal intracellular vesicles with acid phosphatase activity. The basal labyrinth of the lower tubule exhibits considerable alkaline phosphatase activity which is apparently identical in location to the enzyme revealed by two different ATPase localisation techniques.The authors are indebted to the Science Research Council for financial supportThe authors wish to thank Mrs. Margarita Petri for her technical assistance and advice     

A microfluorometric method for alkaline phosphatase: Application to the various segments of the nephron

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.     

Differential effects of temperature on a membrane adenosine triphosphatase and associated phosphatase.

Arrhenius plots of a membrane (Na+ + K+)-dependent ATPase (adenosine triphosphatase) activity showed characteristic discontinuities, whereas those of the associated K+-dependent phosphatase activity did not. These findings support the contention that the phosphatase activity does not depend on phospholipid in the same way as does the ATPase activity.     

Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.

Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K⁺. K⁺-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K⁺ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

     

Presence of ATPase and alkaline phosphatase activities in the starfish sperm acrosome

ATPase activity was cytochemically detected in the peripheral acrosomal component of ionophore-reacted sperm, while alkaline phosphatase activity was demonstrated in the upper and central components of the acrosome and, at fertilization, at the site of sperm-oocyte binding. Supernatants of ionophore treated sperm suspensions were assayed for ATPase, alkaline and acid phosphatase activities. Results suggest that alkaline phosphatase may be involved both in the acrosomal reaction and oocyte jelly lysis but the function of the acrosomal ATPase remains unknown.     

Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase.

The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship.     

Structure and mechanism of the phosphotyrosyl phosphatase activator

Phosphotyrosyl phosphatase activator (PTPA), also known as PP2A phosphatase activator, is a conserved protein from yeast to human. Here we report the 1.9 A crystal structure of human PTPA, which reveals a previously unreported fold consisting of three subdomains: core, lid, and linker. Structural analysis uncovers a highly conserved surface patch, which borders the three subdomains, and an associated deep pocket located between the core and the linker subdomains. The conserved surface patch and the deep pocket are responsible for binding to PP2A and ATP, respectively. PTPA and PP2A A-C dimer together constitute a composite ATPase. PTPA binding to PP2A results in a dramatic alteration of substrate specificity, with enhanced phosphotyrosine phosphatase activity and decreased phosphoserine phosphatase activity. This function of PTPA strictly depends on the composite ATPase activity. These observations reveal significant insights into the function and mechanism of PTPA and have important ramifications for understanding PP2A function.     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

Cytochemical studies on the localization of alkaline phosphatase and HCO-3-activated adenosine triphosphatase in the brush border membrane of rat duodenal enterocytes

Cytochemical techniques were used to demonstrate, with appropriate controls, alkaline phosphatase and HCO-3-activated adenosine triphosphatase (ATPase) in rat duodenal brush border microvillus membranes. Intense activity of ecto-alkaline phosphatase activity was demonstrated with 2-glycerophosphate as substrate. Although biochemical assays suggested that L-phenylalanine inhibited both alkaline phosphatase and HCO-3-activated ATPase, cytochemical studies indicated that there was marked inhibition of alkaline phosphatase revealing a specific HCO-3-activated ATPase on the inner aspect of the microvillus membrane. While it is tempting to suggest that this HCO-3-activated ATPase is implicated in active bicarbonate secretion by the duodenum, decisive identification is not yet possible.     

Purification and fluorometric assay of proteinase A from yeast

A kinetic assay system which provides reliable measurements of Na-K-ATPase activity on 0.2 to 0.5-mm segments of renal proximal convoluted tubules isolated from collagenase-digested renal cortical slices is described. The use of collagenase digestion provides higher values for Na-K-ATPase, possibly by making the enzyme more accessible to the reaction system. The advantages of a kinetic vs an endpoint assay include the ability to use the same tubule as its own reference for the determination of total, ouabain-sensitive, and ouabain-insensitive ATPase activity. In addition, it allows dose-response studies on the effect of inhibitors on ATPase activity in the same tubule segment.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo. Acid phosphatase activity of the epitheloid cells in the glomerular afferent arterioles

Histochemical techniques for acid phosphatase activity applied to kidney tissue of the toad Bufo bufo demonstrate that a high enzyme activity is present in the dense granules of the proximal tubule cells, but also in the media cells in the wall of the glomerular afferent arterioles. The acid phosphatase activity is confined to the characteristic granules in these juxtaglomerular cells, which therefore are lysosomal in nature.     

Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue

We have studied the distribution of histochemically detectable alkaline phosphatase in cultures of seminiferous tubule fragments and of peritubular cells from prepubertal rats. The same material also was immunohistochemically evaluated for the presence of desmin-containing intermediate filaments. The comparative analysis of alkaline phosphatase and desmin positivity shows that alkaline phosphatase histochemistry selectively detects desmin-containing contractile cells in tubular and peritubular cell cultures. We propose alkaline phosphatase as a novel marker for myoid cells that can be of help in screening, defining, and eventually standardizing the exact composition of peritubular cell cultures, a model that is of increasing interest in the study of cellular interactions in the testis.