A novel phosphatase upregulated in Akp3 knockout mice

Reexamination of the Akp3(-/-) mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3(-/-) gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner (gIAP) as opposed to duodenum-specific IAP (dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant (k(cat)) and a 30% drop in K(m), leading to a 4-fold reduction k(cat)/K(m) compared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3(-/-) mice. Developmentally, Akp3 expression is turned on during postnatal days 13-15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3(-/-) mice fed a high-fat diet.     

Purification of Primordial Germ Cells from TNAPMouse Embryos Using FACS-gal

Tissue nonspecific alkaline phosphatase (TNAP), the product of theAkp2locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with theAkp2^tm1Sormutant allele of TNAP expresslacZ(β-galactosidase; β-gal) under control of theAkp2locus. PGCs were purified fromAkp2^tm1Sorembryos using fluorescence activated cell sorting of β-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kitantibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.     

Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.     

鸡的血型研究 Ⅹ.我国11个地方鸡种的血型和血浆蛋白质多态分析

对我国11个地方鸡种1087羽鸡群进行了血型和血浆蛋白质多态7个位点22个等位基因的群体遗传学分析。结果表明,血型A、C位点,基因频率各鸡种存在显著差异,B位点则没有显著差异,血型和血浆蛋白质多态位点基因纯合系数普遍较低,血型因子分布在群体中较为分散,表明这些地方品种存在较大的选择潜力。血浆蛋白质多态位点,中国与外国的某些鸡种比较,存在着较大的相同或相异,可能意味着我国和外国鸡种起源上的同缘或品种起源、进化上的多样性。聚类分析表明,这11个地方鸡种可系统聚类成4个类群,其结果与目前人们对这些鸡种的表征分类不尽一致。     

Disruption of the murine intestinal alkaline phosphatase gene Akp3 impairs lipid transcytosis and induces visceral fat accumulation and hepatic steatosis

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.     

金属离子和脲对白蜡虫碱性磷酸酶的影响

各种金属离子及脲对白蜡虫Ericerus pela (Chavannes)碱性磷酸酶的活性有不同的影响。从白蜡虫雌成虫中分离纯化得到碱性磷酸酶,加入各种不同浓度的金属离子及脲测定酶的活力。一价金属离子Na⁺、K⁺、Li⁺对酶活力没有影响。碱土金属离子Ca²⁺、Mg²⁺、Ba²⁺对酶有激活作用,激活作用的大小顺序依次为Ca²⁺、Ba²⁺、Mg²⁺。第一过渡金属离子中,Mn²⁺、Co²⁺、Ni²⁺对酶有激活作用,而Zn²⁺、Cu²⁺有抑制作用。重金属离子Cd²⁺、Pb²⁺对酶有抑制作用。Ca²⁺激活作用表现为非竞争性激活效应。Cu²⁺抑制作用表现为非竞争性抑制效应。脲对碱性磷酸酶有变性失活作用,按脲浓度可分为低于3 mol/L和高于3 mol/L两种类型。低浓度的脲对白蜡虫碱性磷酸酶的活性抑制的动力学表现为混合型效应。     

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.     

Ablation of TNAP function compromises myelination and synaptogenesis in the mouse brain

Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in skeletal and dental hypomineralization and severe neurological symptoms. TNAP is expressed in the synaptic cleft and the node of Ranvier in normal adults. Using TNAP knockout (KO) mice (Akp2(-/-)), we studied synaptogenesis and myelination with light- and electron microscopy during the early postnatal days. Ablation of TNAP function resulted in a significant decrease of the white matter of the spinal cord accompanied by ultrastructural evidence of cellular degradation around the paranodal regions and a decreased ratio and diameter of the myelinated axons. In the cerebral cortex, myelinated axons, while present in wild-type, were absent in the Akp2 ( -/- ) mice and these animals also displayed a significantly increased proportion of immature cortical synapses. The results suggest that TNAP deficiency could contribute to neurological symptoms related to myelin abnormalities and synaptic dysfunction, among which epilepsy, consistently present in the Akp2 (-/-) mice and observed in severe cases of hypophosphatasia.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

A cell culture system is characterized for monolayers of immature rat epididymal epithelial cells grown on permeable supports. Cover of the filters was achieved by days 4-5 and was maintained for 9-12 days. The secretion of acid phosphatase (ACP), alkaline phosphatase (AKP) and N-acetylglucosaminidase (NAG) into apical and basal compartments of culture chambers was monitored with time in culture for cells from the proximal and distal epididymis of 37-day-old animals. There was independent secretion of the three enzymes: secretion of NAG and AKP was mainly apical, that of ACP basal; daily secretion of ACP and AKP was constant throughout culture, that of NAG declined; there was greater secretion of NAG and AKP by cells from the proximal than the distal region. The initial high apical secretion of NAG is thought to reflect loss of enzyme from unattached cells, whereas the later AKP secretion is truly directional. Secretion was not influenced by the enzymes used in cell preparation. The cytotoxic agent Thimerosal inhibited secretion of all enzymes when placed beneath the cultures, indicating that secretion depended on viable cells, but initially stimulated release of AKP when applied above the cells possibly reflecting release from the cell membrane.     

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.     

重金属镉对鲫鱼碱性磷酸酶和酸性磷酸酶活性的影响

研究了重金属镉对鲫鱼肠、肝胰脏、鳃组织碱性磷酸酶和酸性磷酸酶活性的影响。结果表明,在0.2、0.4、0.8mg/L镉浓度条件下静态染毒12h、24h、48h、96h后,鲫鱼肠、鳃组织中碱性磷酸酶(AKP)和酸性磷酸酶(ACP)的活性降低,肝和胰脏的碱性磷酸酶活性没有明显变化,其酸性磷酸酶活性则升高。     

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b, k) and controlled by a gene locus closely linked to theAkp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i. e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78 000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing theLy-31 andAkp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.     

Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.     

Phosphoprotein Phosphatase Activities in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of τ to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated τ could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of τ might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used ³²P-labeled phosphorylase kinase and poly(Glu.Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/ phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of τ in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of β-amyloid by augmenting the amyloidogenic pathway processing of β-amyloid precursor protein.