Enzymatic and immunological properties of alkaline phosphatase of bullfrog

Enzymatic and immunological properties of alkaline phosphatase (ALPase) in several tissues of bullfrog (Rana catesbeiana) were investigated. Inhibition and thermal inactivation studies showed that bullfrog ALPases in kidney, liver, and intestine had similar enzymatic properties. In addition, mouse antiserum against bullfrog liver ALPase cross-reacted with kidney and intestine enzymes as well as with liver enzyme. These results suggest that a single phenotype of ALPase exists in all tissues of bullfrog in contrast to two or three isoenzymes in mammals.     

Detection of minor immunological differences among human "universal-type" alkaline phosphatases

Two clones of monoclonal antibodies against swine alkaline phosphatase (ALPase; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1), which were useful in distinguishing human kidney and bone ALPases from liver ALPase, were successfully raised in mice. On the other hand, polyclonal antibody cross-reacted not only with human kidney ALPase but also with all other human universal type ALPases. The difference in cross-reactivity of monoclonal and polyclonal antibodies may be caused by the specific antigenicity of human enzymes. The monoclonal antibodies were able to recognize minor heterogeneity that could not be distinguished by their enzymatic properties. The present monoclonal antibody preparations will be utilized for clinical as well as basic investigations to detect minor heterogeneity among universal-type ALPases.     

The rabbit differs from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes

There are only two gene loci code for alkaline phosphatase of mammalian other than human and great apes: one for the intestinal form and other for the liver/kidney/bone form. The former form is present only in the intestine and the latter form occurs in other tissues such as liver, kidney and bone. In the present study, the rabbit was found to be different from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes: only in the rabbit, most of the enzyme in the kidney and liver was the third form which differs from the liver/kidney/bone form, and this form was enzymatically and immunologically similar to the intestinal form of ALPase.     

Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization.

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.     

Increase of Specific Activity, Electrophoretic Type-Transition and Gene Expression of Alkaline Phosphatase during Endodermal Differentiation of F9 Mouse Embryonal Carcinoma Cells

In F9 mouse embryonal carcinoma cells, the specific activity of alkaline phosphatase (ALPase) increases markedly during endodermal differentiation induced by retinoic acid (RA) treatment, but the specific 5'-nucleotidase activity of a similar ecto-phosphatase increases only temporally. Polyacrylamide disc gel electrophoresis showed that F9 cells express only type I ALPase, whereas RA-treated F9 cells express both type I and type II ALPases. Type II ALPase is a minor form on day 1 of RA treatment and becomes the major form on day 4. RA-treated F9 cells also expressed mRNAs for endoderm cell-specific molecules, such as α-fetoprotein, type IV collagen and laminin B1 chain, but their expression of M₂-type pyruvate kinase mRNA of an essential non-ectoenzyme remains constant throughout endodermal differentiation. Northern blot analyses showed that type I ALPase was encoded by a liver (L)/bone (B)/kidney (K)/placenta (P)-type mRNA. The expression of L/B/K/P-type ALPase mRNA was induced in RA-treated F9 cells, but its increase preceded that of ALPase specific activity. These results suggest that the expression of L/B/K-type ALPase is regulated at the translational and/or post-translational level. The differential inhibition of ALPases by L-phenylalanine/L-homoarginine and the thermal inactivation (56°C for 60 min) inferred that type II ALPase was also an L/B/K-type isozyme.     

DEMONSTRATION OF ALKALINE PHOSPHATASE PARTICIPATION IN THE MINERALIZATION OF OSTEOBLASTS BY ANTISENSE RNA APPROACH

MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralizationof ECM.     

Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes

Summary Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES¹ buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES² or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration.In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.1 PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - 2 HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid This study was supported by a Grant-in Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture, the Japanese Government (No. 57770012)     

Translocation of intestinal alkaline phosphatase in streptozotocin-induced diabetic rats

1. We determined the organ of origin and possible mechanism of translocation into the circulation of alkaline phosphatase (ALPase) in the diabetic rat. 2. Experimental diabetes was induced by injection of streptozotocin, resulting in a 8.2-fold elevation in serum ALPase activity. In this case, the major ALPase isozyme detected in serum was intestinal ALPase. 3. In in vitro experimental systems, ALPase was readily released from the duodenal plasma membrane by bacterial phosphatidylinositol-specific-phospholipase C (PI-PLase C) but little if any was released from the ileal membrane. 4. Serum and ileal ALPases were identical in terms of molecular size, whereas duodenal ALPase clearly differed from the serum enzyme. 5. Based on an investigation of the sugar moiety, more of the fraction having higher concanavalin A affinity was found in serum ALPase than with in the case of either of the intestinal ALPases. Serum and intestinal ALPases also differed slightly regarding isoelectric points. 6. Consequently, these data suggest that the serum ALPase of the diabetic rat is derived from ileal ALPase, and it is unlikely that the appearance of ALPase in the circulation is simply the result of solubilization by the action of PI-PLase C or phospholipase D.     

Isolation of GnafC, a polysaccharide constituent of Gnaphalium affine, and synergistic effects of GnafC and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells

After screening extensively factors in plant extracts that increase alkaline phosphatase activity, an osteoblastic differentiation marker protein in mouse calvarial osteoblast MC3T3-E1 cells, GnafC derived from Gnaphalium affine, was found to significantly enhance the alkaline phosphatase (ALPase) activity in a synergistic manner with ascorbate. GnafC was a polysaccharaide with an approximate molecular mass of 10,000 and comprised mannose, xylose, arabinose, galactose and glucose in a molar ratio of 1:2:4.3:2.5:2.7. Expression of the osteoblastic differentiation marker genes was examined by semiquantitative RT-PCR with RNAs prepared from cells at different developmental stages. With ascorbate in the culture, GnafC enhanced the expression of the ALPase and MMP13 genes from the early stage of differentiation, leading to maturation of the collagenous extracellular matrix (ECM), a prerequisite for mineralization.     

Alkaline phosphatase-positive reticular cells of chicken bone marrow--in vivo and in vitro studies

We examined alkaline phosphatase (ALPase)-positive reticular cells from chicken bone marrow in vitro in relation to other varieties of adherent cells. ALPase activity was found in both reticular and adipose cells which formed epithelioid cell colonies and were negative for fibronectin. We observed transition between two cell types. ALPase-negative macrophages as small round cells in culture revealed positive fibronectin and transformed into ALPase-negative spindle cells in long-term culture. Thus, we suggest two cell lineages, each with distinct cell characteristics.     

A Cytochemical Study of ACPase and ALPase Activity in the Laminar Hair Cells of Bresenia shreberi

The ultrastructural distribution of acid phosphatase (ACPase) alkaline phosphatase (ALPase) and the changes of their activities in the laminar hair cells of Bresenia shreberi Geml. were studied by means of lead phosphate preciptation method during their development. The ACPase activity became detactable at the stage of mucilage secretion of the head cell, which was located at endoplasmic reticulum (Er),cuticle (Cu) and tonoplast (Tp) of the head cell, and nuclear membrane (NM), plasmodesmata (P1) and wall ingrowths (WI) of the stalk cell, and WI and plasmolemma of basal cell. ALPase activity showed its first appearance at the mitochondria(Mi) of the basal cell at the stage of head cell formation. At the stage of head cell secretion, ALPase activity was located at Er, Cu, Mi of head cell, and NM, Go, Er, WI and P1 of stalk cell. ALPase activity at Mi of basal cell was most intensive. The results indicated that ACPase and ALPase possessed the function of promoting cell differentiation and material transfer in the process of hair cell development and that the material for synthesizing mucilage in head cell was provided by the basal cells and the parenchyma cells nearby.     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Covalent protein immobilization on glass surfaces: application to alkaline phosphatase

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4+/-0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。       

Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: Evidence for a difference between human parathyroid hormone-related protein and human parathyroid hormone

We have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast-like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone-related protein (hPTHrP). Pretreatment of human osteoblast-like SaOS-2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1-34) but had no effect on the actions of hPTHrP(1-34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L-Phe-Gly-Gly, an inhibitor of hpALPase. Treatment of SaOS-2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS-2 cells. In rat osteoblast-like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1-34) or hPTHrP(1-34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS-2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase. © 1994 Wiley-Liss, Inc.     

Alkaline phosphatase activity in the placental labyrinth of the cat and problems of specific localization of transport adenosine triphosphatase. An ultrastructural study (author's transl)

The ultrastructural localizations of alkaline phosphatase (ALPase) and of Na+-K+-dependent adenosine triphosphatase (Na+-K+-ATPase) were studied in the placental labyrinth of the cat during the last days of gestation. ALPase activity could be detected in the syncytiotrophoblast but was absent from maternal tissues. Enzyme activity was observed only along plasma membranes of microvilli and absorption tubules on the maternal surface of the syncytium and also on the podocytes-like cytoplasmic processes of the fetal face. The localization of the Na+-K+-ATPase activity as obtained with the method of Ernst was identical with that of ALPase. This activity was not very ouabaine sensitive or K+ dependent, but was almost completely inhibited by levamisole. The strong ALPase activity of the syncytiotrophoblast does not allow a specific detection of Na+-K+-ATPase. However, the localization of these enzymes activities on syncytiotrophoblast surfaces directly related to fetal and maternal capillaries could suggest that these surfaces are associated with transport mechanisms of the trophoblast.     

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.