Biochemical characterization of alkaline phosphatase in guinea pig thymus.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.     

Cholinergic-stimulated alkaline phosphatase secretion and phospholipid synthesis in guinea pig seminal vesicles

Incubation of slices of seminal vesicles of the guinea pig with cholinergic drugs led to an enhanced secretion of alkaline phosphatase. Incubation with carbamylcholine also stimulated the incorporation of P³² into the phospholipid fraction. Both cholinergic effects required a supply of energy since dinitrophenol was inhibitory. The stimulation of enzyme secretion and phospholipid synthesis by carbamylcholine was completely abolished by atropine. Omission of calcium ions from the incubation medium and pre-treatment of the slices with ethylenediaminetetraacetic acid (EDTA) caused a marked reduction in alkaline phosphatase secretion induced by carbamylcholine but had no effect on incorporation of P³² into phospholipids. Adenergic agents such as epinephrine, norepinephrine and isoproterenol did not influence these two processes. Addition of cyclic AMP, dibutyryl cyclic AMP and a phosphodiesterase inhibitor was also ineffective. The incorporation of P³² into the various phospholipids of the seminal vesicle was examined. In the presence of carbamylcholine, there was a marked increase in the P³²-specific activities of phosphatidylinositol (nearly 6-fold) and of phosphatidylserine (about 1.5-fold). These observations indicate that the guinea pig seminal vesicle, a large hollow organ composed of a single layer of epithelium, is ideally suited for studies concerning the biochemistry of macromolecular secretion.     

Polymorphic patterns of heterochromatin distribution in guinea pig chromosomes

The chromosome complement and patterns of heterochromatin distribution (as demonstrated by the DNA d-r method) were studied from three different guinea pigs. Karyotype analyses showed that one of the females had a heteromorphic sex pair formed by a submetacentric X chromosome and a subterminal X chromosome originated by a shortening of the short arm (x-chromosome). The heterochromatin was mainly found in the pericentromeric areas of the autosomes and X chromosomes and in the short arm of pair 7. The Y chromosome exhibited a degree of heterochromatinization different from that of pericentromeric areas.—The analysis of the heterochromatin distribution in the X chromosomes showed that the smaller size of the heteromorphic x-chromosoine was probably due to a lack of heterochromatin in its short arm. Moreover, two out of the three animals studied had a heteromorphic pattern of heterochromatinization in the pair 21 characterized by heterochromatinization of the pericentromeric area in one chromosome and almost complete heterochromatinization of the other homologue.—It is suggested that most of the heterochromatin disclosed by the DNA d-r method is formed by repetitious DNA; and that the Y chromosome and perhaps some autosome regions in guinea pigs are formed by a type of heterochromatin with properties different from those of the constitutive and facultative heterochromatin (intermediate heterochromatin).Supported in part by NIH Grant 5-501-RR05672-02 and by NIH contract 70-2299.     

Localization of alkaline phosphatase and NADH diaphorase in the principal cells of the guinea pig epididymis

The activities of alkaline phosphatase and reduced nicotinamide adenine dinucleotide (NADH) diaphorase in the principal cells of the guinea pig epididymis were studied histochemically. Alkaline phosphatase activity was absent from the principal cells but was present in the basement membrane of the epididymal epithelium. NADH diaphorase activity was distributed throughout the cytoplasm of the principal cells in each epididymal segment. There was a gradual increase in NADH diaphorase activity from segments 1 through 7. Possible functions of alkaline phosphatase and NADH diaphorase in the epididymis are discussed.     

Fas modulates both positive and negative selection of thymocytes.

We studied the functional role of Fas (CD95) in thymic T cell development using the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice. In DO10 lpr/lpr mice, the differentiation of CD4(+)CD8(+) double-positive (DP) thymocytes to CD4(+) single-positive (SP) thymocytes was markedly impaired, as indicated by decreased generation of CD4(+) SP thymocytes and reduced ratio of CD4(+) SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of +/+ mice. Activation of DP thymocytes in the process of positive selection was also significantly inhibited in DO10 lpr/lpr mice, as shown by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to +/+ mice. Furthermore, the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 micrograms) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 +/+ mice. On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and +/+ mice. Together, these results indicate that Fas is importantly involved in both positive and negative selection of thymocytes.     

Catalytic properties of alkaline phosphatase from pig kidney

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.     

Placental calcium and phosphorus transfer in the guinea pig: lack of effect of modulators of Ca-ATPase and alkaline phosphatase activity

The effects of modulators of Ca-ATPase and alkaline phosphatase (AP) activity on placental calcium and phosphorus transfer were studied using the in situ perfused guinea pig placenta. The diuretics ethacrynic acid and furosemide had no significant effect on placental calcium and phosphorus transfer when injected into the mother (1.0 or 10.0 mg X kg-1) or added to the solution perfusing the fetal side of the placenta (0.25 or 2.0 mM). These two drugs have previously been shown to inhibit placental Ca-ATPase and enhance AP activity in vitro. D-Penicillamine, which inhibits placental AP but not Ca-ATPase activity in vitro, also had no significant effect on net calcium and phosphorus transfer from mother to fetus either when given to the mother (50 mg X kg-1) or added to the placental perfusion solution (0.25 or 2.0 mM). These results suggest that placental transfer of calcium and phosphorus in the guinea pig may not be directly related to placental Ca-ATPase and AP activities.     

Schistosoma mansoni: distribution and characteristics of alkaline and acid phosphatase

The biochemical distribution and characteristics of the alkaline and acid phosphatase activities in both sexes of Schistosoma mansoni are reported. Alkaline and acid phosphatase activities were found in the epidermis as well as in the internal tissues. Alkaline activity is mostly located within the epidermis in both sexes. The acid activity is high in the epidermis of males and more or less equally distributed in females.Starch gel electrophoresis of worm homogenates revealed two sites of activity for both phosphatases. One is an anodic band which is a soluble phosphatase, the other, a membrane bound phosphatase, which remains at the origin under the electrophoretic conditions used. These findings are discussed in relation to previous results and to the possible roles played by these enzymes in adult S. mansoni.     

Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.     

Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines

Morphologically macrophage-like cells were cloned from hamster bone marrow cells by coculturing bone marrow cells with hamster chondrocytes. One of the clones (CCP-2) was characterized in the present study. CCP-2 cells were positive in an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and non-specific esterase (NSE). We showed CCP-2 cells degraded cartilage matrix and hydroxyapatite coated on Osteologic disks. A gelatinase secreted from CCP-2 cells was observed and purified from serum-free conditioned medium of the cells. N-terminal amino acid sequencing of the purified enzyme revealed it was matrix metalloproteinase-9. However, CCP-2 cells failed to express calcitonin receptors, a mature osteoclast marker, even after coculture with osteoblast ST2 cells in the presence of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3]. The cells showed high affinity to types X and I but not to type II collagen. In addition, histochemical studies have shown the presence of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cells at the secondary ossification site of the hamster humerus. From these observations, we concluded that CCP-2 cells are similar to osteoclast but not the same. CCP-2 cells are therefore important tools for investigating chondroclastogenesis/osteoclastogenesis and endochondral ossification.     

Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme.

The highest total activity of estrogen sulfotransferase in guinea pig is in liver and the highest specific activities are in the adrenal and the midgestational chorion. Guinea pig gonads contain scarcely detectable activities. In CD-1 mice the highest specific activity is in testis and the highest total activity is in late placenta. Adrenals from both sexes and ovaries contain minimal activities, while liver and fetal membrane activities are remarkably low. In CD-1, DBA, C57BL, and BALB mice, qualitative patterns are similar. Purified or partially purified estrogen sulfotransferase from guinea pig adrenal and chorion were used to study the effect of a number of possible steroidal inhibitors. Considerable structural specificity is evident within a range of steroids, even among some which are not substrates. Pregnenolone is the most effective 21-carbon inhibitor and, in general, more highly hydroxylated forms are less inhibitory. Within a series of 21-, 19- and 18-carbon steroids, the structure of the A ring appears to be extremely important in regard to inhibitory effects.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.