NADPase Activity Research Articles

Three-dimensional morphology of the Golgi apparatus in osteoclasts: NADPase and arylsulfatase cytochemistry, and scanning electron microscopy using osmium maceration.

This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 μm or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus.

MJ0917 in Archaeon Methanococcus jannaschii Is a Novel NADP Phosphatase/NAD Kinase

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.

Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: implication in regulation of NAD+/NADP+ balance.

NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+-degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7-8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5'-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol.

Changes of cytochemical properties in the Golgi apparatus during in vivo differentiation of the ameloblast in developing rat molar tooth germs.

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activities were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcompartments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.

Ultrastructural Distribution of NADPase within the Golgi Apparatus and Lysosomes of Mammalian Cells

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural localization of CMPase, TPPase, and NADPase activity in neurons, satellite cells, and Schwann cells in frog dorsal root ganglia.

Sections of bullfrog dorsal root ganglia were analyzed for cytidine monophosphatase (CMPase), thiamine pyrophosphatase (TPPase), and nicotinamide adenine dinucleotide phosphatase (NADPase) activity, and the distributions of these enzymatic activities were compared with those traditionally found in other cell types (e.g., CMPase: Golgi trans-sacculotubular network; TPPase: trans-Golgi saccule(s); NADPase: intermediate Golgi saccules). In the present study, CMPase activity in neurons was localized mainly to the Golgi trans-sacculotubular network and lysosomes, but sometimes also occurred at the ends of the trans and most distal intermediate Golgi saccules. A similar distribution was found in satellite and Schwann cells. TPPase activity in neurons occurred not only in the trans-Golgi saccule but also in the trans-sacculotubular network, lysosomes, and scattered tubular elements. In satellite and Schwann cells, activity was found in both the trans saccule and trans-sacculotubular network, and substantial activity often appeared in the more distal of the intermediate saccules. NADPase activity in neurons was usually absent from the intermediate Golgi saccules and was confined to the trans-sacculotubular network and lysosomes; however, activity was sometimes also found in the intermediate and/or trans-Golgi saccules. In satellite and Schwann cells, activity appeared consistently in both the trans-sacculotubular network and intermediate saccules, as well as in lysosomes. These distributions, especially in the case of TPPase and NADPase, differ substantially from the most frequently reported localizations of the above enzymes, indicating that the Golgi complex may exhibit considerable plasticity of structure and function in different cell types.

Cytochemical localization of nicotinamide adenine dinucleotide phosphatase(NADPase) and nucleoside diphosphatase activities in the Golgi apparatus of the rat epididymis.

The ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) and thiamine pyrophosphatase (TPPase) or nucleoside diphosphatase (NDPase) activities was studied in the principal cells of the rat epididymis and compared with the pattern produced after monensin treatment.The NADPase activity was found positive in the intermediate cisternae near the cis side of the Golgi lamellae, to show a difference in compartment from that of TPPase on the trans side. After 60min treatment with monensin, most of the Golgi stacked lamellae exhibited dilation. The swelling occurred in all stacks without preferential effect on any particular Golgi subcompartment. With these enzymes, the characteristic staining patterns on the subcompartments in the Golgi apparatus were basically unchanged after treatment with monensin. However, some of the NADPase positive intermediate cisternae on the cis side of the Golgi apparatus were remarkably dilated.

Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver.

We examined the distribution of acid NADPase activity in rat hepatocytes by cytochemistry and subcellular fractionation. Cytochemical studies revealed strong NADPase activity in many lysosomes and Golgi saccules situated between the medial and trans aspect of the stack. Light, spotty deposits of reaction product also were observed frequently within elements of the Golgi-associated endoplasmic reticulum lysosome (GERL) system. Biochemical studies with liver homogenates indicated that NADPase activity was distributed across various subcellular fractions in a pattern resembling typical nonspecific acid phosphatase activity, as monitored using beta-glycerophosphate as substrate. Thus, by differential centrifugation, most cellular NADPase activity was found in the large granule (ML) fraction (61%). The microsomal (P) fraction showed about 17% whereas the nuclear (N) and cytosol (S) fractions each accounted for about 11% of the remaining NADPase activity, respectively. Separation of the ML fraction into endosomes and secondary lysosomes revealed most of the acid NADPase activity associated with lysosomes. An alternate one-step method for isolation of fractions indicated that 3% of the recovered acid NADPase activity, but 55% of the Golgi marker enzyme galactosyl transferase, was associated with a purified intact Golgi fraction. Cytochemical studies with ML and intact Golgi fractions indicated that bona fide reaction product was localized to lysosomes and to the same saccules that were reactive in vivo. We propose that saccules making up the trans half of the Golgi stack may represent a site for accumulation of a biogenetic precursor of the lysosomal acid NADPase in hepatocytes.

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity within columnar, goblet, and Paneth cells of rat small intestine.

The distribution of nicotinamide adenine dinucleotide phosphatase (NADPase) activity was examined in epithelial cells of rat small intestine. Segments of ileum were fixed with glutaraldehyde and tissue chopper sections were incubated for up to 4 hr at pH 5.0 in cytochemical media prepared with NADP as substrate. NADPase activity was found primarily within the Golgi saccules of columnar, goblet, and Paneth cells. Columnar and goblet cells showed most of the NADPase activity within the saccules which were intermediate between the cis and trans faces of the Golgi stack. Paneth cells, however, showed the heaviest staining within saccules between the intermediate and innermost saccule at the trans aspect of the Golgi stack. Both columnar cells and Paneth cells also contained spotty, and sometimes heavy, deposits of reaction product within an occasional focal area of the GERL system and within an occasional lysosome. Control experiments indicated that the Golgi-associated NADPase activity was enhanced if cells were pretreated for about 12 hr with EGTA prior to incubation. No similar enhancement was apparent if the tissues were pretreated with DMSO. Furthermore, NADPase activity within the Golgi saccules could be inhibited completely by incubating intestinal epithelial cells with NADP in the presence of sodium fluoride or L(+)-tartrate.

A complex network of “snake-like tubules” revealed by the NADPase cytochemical reaction in the acinar cell of pancreas

We have recently described the ultrastructural localization of NADPase activity in the exocrine pancreas of rat. The enzyme was found in the intermediate saccules of the Golgi apparatus, in dense bodies and lysosomes but was absent from zymogen granules. A very intense reaction was noticed in a peculiar structure which was termed “Snake-Like Tubule” (SLT). The purposes of the present study were firstly to delineate SLT distribution in the acinar cell and secondly to define any possible relationship or association with other cellular organelles.NADPase cytochemical reaction was performed on the pancreas of adult Sprague Dawley rats. Small lobules were excised and fixed for 50 min, at 4°C, in 2% glutaraldehyde buffered with 0.1M cacodylate at pH 7.2. Lobules were rinsed several times with the same buffer containing 570 sucrose and cut with a Mcllwayn tissue chopper. Sections were washed several times with buffer and incubated for 2 hr at 37°C in the following medium: 4mM NADPH; 40mM sodium acetate buffer, pH 5.0; 4mM lead acetate and 5% sucrose.

Nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate splitting enzyme(s) of sheep and rabbit erythrocytes: their effect on the growth of Haemophilus.

Fragility of rabbit erythrocytes in agar plates results in gradual release of their NAD and NADP contents into the medium. Due to high NADase and negligible NADPase activity of rabbit red blood cell stroma at neutral pH, the NAD released into the medium is hydrolyzed and NADP remains intact. Thus, rabbit erythrocytes and their lysates support the growth of NAD(P)-requiring Haemophilus by serving as a source of NADP. Stability of sheep erythrocytes in agar plates results in retention of their NAD and NADP contents and consequently in inhibition of growth of NAD(P)-requiring Haemophilus. The highly active NAD- and NADP-splitting enzyme(s) of sheep red blood cell stroma prevent(s) the growth of Haemophilus on sheep blood lysates through inactivation of both NAD and NADP which are released into the medium.

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity to the intermediate saccules of the Golgi apparatus in rat incisor ameloblasts.

Cytochemical evidence for the existence of a Golgi-associated phosphatase activity that hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) at acid pH in rat incisor ameloblasts was obtained by incubating sections from glutaraldehyde-fixed teeth in a medium containing NADP as substrate and lead ions as capture agent. Following incubation for 1 hr at 37 degrees C and pH 5.0, the Golgi saccules situated between those at the cis (immature) and trans (mature) faces of the ameloblast Golgi apparatus were marked by reaction product with the heaviest deposit in the middle saccule. Reaction product was otherwise seen in trace amounts only over some elements of the GERL system as well as a few lysosomal dense bodies and immature secretory granules. Control experiments established that the selective staining of intermediate Golgi saccules at pH 5.0 could only be duplicated by using substrates that resembled the complete NADP molecule, and not just the portion containing the adenosine 2'-monophosphate group. As well, no deposits of reaction product were seen within the Golgi saccules of ameloblasts incubated at pH 5.0 with nictoinamide adenine dinucleotide (NAD) as the substrate or that were incubated at pH 7.2 or pH 9.0 with NADP as the substrate. It was concluded that a specific, acid-NADPase activity is present in the intermediate Golgi saccules of secretory ameloblasts. Preliminary observations on other cells suggest that the localization of NADPase activity to Golgi saccules may constitute a general phenomenon.

Rabbit erythrocyte pyridinnucleotides and nucleosidase activity after x-ray irradiation.

SummaryThe effect of irradiation (600 R) of rabbit erythrocyte NAD and NADP was studied at various intervals after whole-body irradiation of the animal. The decrease found in both pyridinnucleotides progressed with time. The decrease in NAD and NADP was significant after the first hour of irradiation. In the cells of the irradiated animals an increase was found in NAD-ase and NADP-ase activity, which accounts for the decrease in pyridinnucleotide concentrations. Nicotinamide inhibits the activity of enzymes both in normal and irradiated animal erythrocytes. The protective effect of nicotinamide is more marked on the NADP than on the NAD system.

Effect of NADP-glycohydrolase on glucose-6-phosphate dehydrogenase.

The action of NADPase on glucose-6-phosphate dehydrogenase from human erythrocytes and leukocytes and from the rat liver has been studied. The NADPase was partially purified from rat spleen mitochondria and from erythrocyte stroma. The G-6-P-DH was purified 1450 fold from erythrocytes, 333 fold from leukocytes and 379 fold from rat liver. NADPase added to G-6-P-DH preparations leads to an inactivation which increases as a function of the used concentrations. When equal amount of NADPase are added, both from stroma and from spleen, a similar inactivating effect on G-6-P- DH is obtained. On the other hand no proportionality was obtained between NADPase activity and inactivation of G-6-P-DH; no stronger inactivation than 80% was ever reached. Our results give evidence for the hypothesis that the inactivation of G-6-P-DH is due to the cleavage of the nicotinamide-ribose linkage which binds the two monomers. The presence of NADPHj, as it was found in the enzyme molecule, could explain the non-total inactivation which could he obtained.