The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase

The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

Summary The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.This study was supported by a grant from the Japan Educational Ministry     

Comparative distribution of glutamyl and aspartyl aminopeptidase activities in mouse organs.

To evaluate the functional role of glutamyl and aspartyl aminopeptidases, their soluble and membrane-bound activities were measured simultaneously in several tissues of normal mice using arylamide derivatives as substrates. Although the soluble aspartyl aminopeptidase activity showed its highest levels in the testicle, the rest of the activities presented their highest levels in the kidney. Different patterns of distribution were observed for glutamyl and aspartyl aminopeptidase activities and also for soluble and membrane-bound aspartyl aminopeptidase activities. However no major differences were observed between soluble and membrane-bound glutamyl aminopeptidase activities. This unequal distribution suggests that the use of arylamide derivatives as substrates is a sensitive method that distinguishes between these enzymatic activities. The results also suggest different functions for soluble and membrane-bound aspartyl aminopeptidase activities, and for glutamyl and aspartyl aminopeptidase activities.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.     

Effects of AET and MEA on the lysosomal hydrolase activities in the mouse organs.

The adult male Swiss mice were injected intraperitoneally with AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl), in a toxic dose of 400 mg/kg body weight. The acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E. C. 3.1.6.1) activities in crude homogenates of liver and kidneys were assessed every fourth hour throughout a 24-h period. Different patterns of temporal changes in the acid phosphatase and arylsulphatase activities in liver and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue and per the whole organ weight, were found. The extent and timing of the alterations in the activity of each of the lysosomal hydrolases were dependent on the particular organ chosen and aminothiol compound given.     

Molecular cloning of the mouse lysosomal acid phosphatase

The mouse cDNA for lysosomal acid phosphatase was cloned. The deduced amino-acid sequence shows 89 and 96% identity with that of the human and rat enzyme, respectively. Namely all residues known to be important for the structure, catalytic activity and transport of lysosomal acid phosphatase are conserved among the three species.     

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.