Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts.

The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.     

Correlations between the activities of semen acid phosphatase and Ca(2+)-dependent ATPase and age in different breeds of cocks.

1. The present work aims to find a biochemical criterion for evaluating the evolution of sperm according to age through the study of the ATPase activity from the spermatozoa and the acid phosphatase from the seminal plasma of cocks from three different breeds. 2. The optimal parameters of action of the cock semen acid phosphatase and the Ca(2+)-dependent ATPase from the spermatozoa were studied. 3. The substrate specificity of the semen acid phosphatase and its inhibition by tartrate, fluoride, metavanadate, molybdate and Hg2+ were also studied. 4. The two enzymes were determined from the Sussex, Golden Cornish and Plymouth Rock breeds at different ages. 5. The data lead to the conclusion that some properties of bird spermatozoa are less influenced by breed while the acid phosphatase activity, secreted in the ductus deferens is a breed characteristic.     

Testicular Ca++ ATPase activity in mice: effects of age and gonadotropin administration

These studies describe a high affinity calcium (Ca++)-dependent ATPase in purified testicular plasma membranes, which exhibits increased activity from weaning age to adulthood. Administration of human chorionic gonadotropin (hCG; 5 IU) increased enzyme activity in 21-day old and pubertal (35 to 40-day old), but not in adult mice. In pubertal mice, these increases in testicular Ca++-ATPase activity were dose-related and evident 60 min after hCG administration. A second challenge dose of 5 IU hCG administered either 24, 48 hrs, or 5 days later, had no additional effect on Ca++ ATPase in purified testicular plasma membranes in these pubertal animals. The present findings indicate that testicular plasma membrane Ca++ATPase activity exhibits a developmental pattern concomitant with increased testicular steroidogenic activity during sexual maturation. Furthermore, enzyme activity is increased by gonadotropic stimulation and exhibits a refractoriness similar to that of androgen biosynthesis to repeated hCG stimulation.     

Evidence for the influence of the protein-phospholipid interface on sarcoplasmic reticulum Ca++ Mg++ ATPase activity.

Sarcoplasmic reticulum from the white hind leg muscle of the rabbit was examined with 31P nuclear magnetic resonance as a nonperturbing probe of phospholipid-protein interactions in the intact membrane. The phospholipids of the sarcoplasmic reticulum appear to inhabit two distinct environments: one very similar in behavior to pure phospholipid lamellar dispersions and the other immobilized by the protein in the membrane. Measurement of the population of the latter environment suggests that it is dependent on salt concentration and probably not due to the Ca++ Mg++ ATPase of the sarcoplasmic reticulum. This immobilization can be removed completely by papain proteolysis of the membrane protein, but only partially by trypsin treatment. The phospholipid composition of recombinants with the Ca++ Mg++ ATPase was varied in order to look for effects of the phospholipid-protein interface on enzymatic activity of the Ca++ Mg++ ATPase. Both transphosphatidylated phosphatidylethanolamine (from egg phosphatidylcholine) and bovine brain phosphatidylserine readily partitioned into the putative boundary layer, whereas under the same conditions soybean phosphatidylethanolamine was excluded. Only phosphatidylserine affected the activity of the enzyme, causing an inhibition that was proportional to the phosphatidylserine content, relative to phosphatidylcholine.     

Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.     

Effects of season and breed on sperm acrosin activity and semen quality of boars

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.     

Isolation and characterization of plasma membranes from the adrenal medulla.

Abstract– The isolation of a plasma membrane fraction from the bovine adrenal medulla and its characterization are described. The plasma membranes are enriched 13-fold in AChE, a plasma membrane marker, and represent 0.7% of the homogenate membrane protein. The yield of these membranes is typically 10-12% by the criterion of the percentage of total membrane bound AChE in the homogenate. The membranes were characterized as to their polypeptide, phospholipid and cholesterol content and compared with chromaffin vesicle, mitochondrial and microsomal membranes by these parameters. Two enzymatic components of the plasma membranes, ATPase and adenylate cyclase, were also studied. Calcium ATPase activity is 2.5-fold higher than magnesium ATPase activity, appears to be the result of a single enzyme, and is a genuine component of the plasma membranes. The magnesium stimulated activity appears to have at least two enzymatic components, one of which may be identical to the calcium ATPase. Adenylate cyclase is a plasma membrane component, but may not be uniquely localized there, as it is rather unstable throughout the fractionation procedure. It is stimulated by GTP (0.7-fold at 10^?5M), GPP(NH)P (4.8-fold at 10^?5M) and sodium fluoride (4.6-fold at 10^?2M). It is refractory to stimulation by all other compounds tested.     

Fine structural localization of adenosine triphosphatase activities in the saccus vasculosus of the rainbow trout,Salmo gairdneri Richardson

The following characteristics of the adenosine triphosphatases (ATPase) in the saccus vasculosus were studied in Salmo gairdneri Richardson: 1) distributional pattern, 2) cytochemical properties in relation to different substrates, inhibitors, pH and bivalent metal ions, and 3) ultrastructural localization. Ultracytochemical studies using modifications of the Washstein-Meisel technique showed that within the pH range 7.1-8.0 several Mg++ or Ca++-activated ATPase are localized on the intracellular surface of membranes and in the cytoplasm of ependymal coronet cells and tanycytes ("supporting cells", "Zwischenzellen", glial cells"). The high ATPase activity at the level of the specialized luminal plasma membranes of coronet cell globules and of tanycyte microvilli is discussed in relation to phenomena of active transport and a possible resulting transfer of low-molecular weight substances into and/or from the cerebrospinal fluid (CSF). The localization of ATPase on the specialized membranes of primary vesicles is considered in connection with available structural and enzyme-cytochemical data on a possible function of these cell organelles in storage and release of substances (including Ca++ ions?). The cytoplasmic ATPase activity in coronet cells is ascribed to microtubules and/or possible existing contractile proteins/filaments, presumably concerned with internal transport or motility processes. In tanycytes ATPase activity is believed to be associated with the characteristic microfilamentous system of still unknown function. The ATPase activity in the (9 + 0) ciliary apparatus of globules could not be interpreted in terms of motility. The present study provides further support to the proposed hypothesis of the transport function of the saccus vasculosus, and an extension of the concept in the sense that not only the principal coronet cells, but also the tanycytes of this circumventricular organ are involved in CSF-homeostasis.     

Detection of a Na+-activated, furosemide-inhibited ATPase activity in rat intestinal mucosa

An ouabain-insensitive, Mg++-dependent, Na+-stimulated ATPase activity which is inhibited by furosemide was found in mucosal homogenate of rat small intestine. The subcellular localization of this ATPase activity was studied by means of isolated purified brush borders and basolateral plasma membranes. The results suggest a nearly identical distribution of Na+-activated and (Na+K+)-activated ATPase within the epithelial cells. Under conditions of alloxan and streptozotocin diabetes an increase of both ATPase activities can be found only in the basolateral plasma membranes. These observations agree well with the convective model of intestinal absorption.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

Cytochemical localization of ouabain-sensitive, K+ -dependent p-nitrophenylphosphatase and Ca++-stimulated adenosine triphosphatase activities in human parotid and submandibular glands

K+ -dependent p-nitrophenylphosphatase (pNPPase) and Ca++ -stimulated adenosine triphosphatase (ATPase) activities were studied in human parotid and submandibular glands using cytochemical methods at the ultrastructural level. In both glands, only the striated-duct epithelium showed K+ -pNPPase reaction product, thereby indicating the localization of Na+, K+ -ATPase. The precipitate was concentrated on the deep invaginations of the basolateral plasma membranes, in close association with their cytoplasmic surface. Ca++ -ATPase activity was also found on the basolateral plasma membranes, but two striking differences from the K+ -pNPPase distribution were observed: firstly, Ca++ -ATPase appeared in both acinar and ductal cells, and secondly, it was localized on the outer side of the plasma membranes.     

Isolation of plasma membranes from ram spermatozoa by a two-phase polymer system

The plasma membranes of ram spermatozoa were disrupted in a hypotonic EDTA medium and isolated by using a two-phase polymer system of dextran--polyethyleneglycol. The plasma membranes obtained were of a relatively high degree of purity (approximately 70%) as judged by electron microscopy observations and measurements of the marker enzymes alkaline phosphatase, ATPase and AMPase. The activity of succinate cytochrome C reductase, a marker of mitochondrial membranes, was very low.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.     

Evidence for nonmuscle myosin in bovine ejaculated spermatozoa

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin-myosin function. An actin-stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized-actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K⁺-EDTA ATPase activity. The sperm actin-myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.     

Identification of an anion-transport ATPase that catalyzes glutathione conjugate-dependent ATP hydrolysis in canalicular plasma membranes from normal rats and rats with conjugated hyperbilirubinemia (GY mutant).

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.     

Acidification activity of human neutrophils. Tertiary granules as a site of ATP-dependent acidification

The acidification activity of human neutrophils, known to occur extracellularly and intraphagolysosomally, was studied in intact and in fractionated cells. The subcellular location of the acidification activity was investigated by rate zonal sedimentation of post-nuclear supernatants from resting cells on continuous sucrose gradients. The acidification measurements indicated a dominance of activity in gelatinase-rich tertiary granules. On the other hand, ATPase activities were located in plasma membrane and in the membranes of the cytoplasmic granules (specific, azurophilic, and tertiary). All of these activities were diminished by the inhibitors dicyclohexylcarbodiimide and diisothiocyanostilbene disulfonic acid; however, studies with other inhibitors, especially N-ethylmaleimide and duramycin, suggested ATPase enzymatic differences depending on location. The results taken together provide direct and strong indication of involvement of a proton pump ATPase in acidification inside neutrophils. Furthermore, the dominant location of acidification activity in tertiary granules that very readily degranulate presumably has significant implications for the importance of low pH in cidal events and the inflammatory process.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

Desensitization of opioid-stimulated GTPase in neuroblastoma x glioma hybrid cells

NG108-15 cells were pretreated with the opioid peptide [D-Ala2, D-Leu5]enkephalin and the opioid-dependent low Km GTPase was assayed in membranes. Pretreatment resulted in a small decrease in basal GTPase activity and led to a concentration-dependent reduction in opioid-mediated stimulation of the enzyme. These effects were observed whether the agonist was present or absent throughout all the experimental procedures, but, in the second condition, the desensitization was smaller. The addition of naloxone had no effect on basal GTPase activity, in either control or pretreated cell membranes. Both Na+ and Mg++ were required for the opioid-induced stimulation of the GTPase. Mg++ enhanced basal enzymatic activity in controls, whereas in membranes from pretreated cells, it produced an inhibition. Thus, desensitization of the opioid-dependent low Km GTPase occurs upon chronic opioid treatment and a Mg++ regulatory site might be altered in the course of this process.