Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Characterization and Purification of Acid Phosphatase from Ancient Human Bone

Abstract

In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10°C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.     

Purification and Characterization of an Acid Phosphatase from Mycoplasma fermentans

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu²⁺, Co²⁺ and Mg²⁺, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-l -tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-l -threonine, O-phospho-l -serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-l -tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Relationship Between Phosphatase Activity and Cytotoxic Effect of Two Protein Phosphatase Inhibitors,Okadaic Acid and Pervanadate,on Human Myeloid Leukemia Cell Line

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km=0.08?mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)—(Km=0.51?mM). Total phosphatase (pNPP) was inhibited in the presence of 10?mM vanadate (98%), 200?μM pervanadate (95%) and 100?μM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24?hours, only 20% residual activity was observed in presence of 200?μM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100?nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100?nM okadaic acid in combination with 200?μM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.     

阿勒泰黄芪提取物对蛋白酪氨酸磷酸酯酶1B的抑制作用

本文探讨了阿勒泰黄芪不同提取物对蛋白酪氨酸磷酸酯酶1B(PTP1B)的抑制作用.采用分光光度法测定了提取物中的黄酮和皂苷含量;通过体外酶促动力学方法检测了不同提取物对PTP1B的影响,并确定了抑制类型;并采用氧化酶法检测了阿勒泰黄芪提取物对细胞利用葡萄糖能力的作用.结果表明,阿勒泰黄芪8种提取物(E1 ～8)中黄酮含量分别为5.09、10.46、3.58、3.23、53.91、21.77、5.76和7.49 mg/mL,其中E1、E2、E6、E7、E8皂苷含量分别为16.53、27.45、21.90、10.21和8.96 mg/mL;各提取物对PTP1B活性均表现出抑制作用,其中E1、E2、E7、E8的IC50分别为34.8、4.7、7.35和7.15 μg/mL,E1、E7和E8是竞争性抑制,E2是混合型竞争性抑制.E1、E2、E5、E7和E8较明显的提高了CHO-K1细胞对葡萄糖的利用.提示皂苷可能是阿勒泰黄芪抑制PTP1B活性的主要物质,通过PTP1B途径有效了提高细胞利用葡萄糖的能力.本研究为阿勒泰黄芪开发为防治糖尿病及改善胰岛素抵抗的药物或保健品提供实验依据.     

有机碳和无机碳对3种真菌胞外酸性磷酸酶和蛋白酶活性的影响

多数研究表明外生菌根真菌能够促进植物养分吸收并提高植物生长,但是对其发生的原因研究较少。本文在室内控制条件下,研究了真菌菌丝分泌N、P相关胞外酶及其受土壤有机碳（胡敏酸）和无机碳（碳酸钙）添加的影响,结果表明：1）3种真菌——松乳菇(Lactarius deliciosus)、变色红菇（Russula integra）、铆钉菇（Gomphidius viscidus）菌丝均能够分泌酸性磷酸酶和蛋白酶,而且多数情况下,MMN培养基培养14 d时,各个酶活性较高,而不同菌的胞外酶活性存在较大的差异,平均值来看铆钉菇酸性磷酸酶活性最低而蛋白酶活性最高,其它2个真菌菌丝的胞外酶活性差异不大;2）添加胡敏酸后,3种菌丝的酸性磷酸酶活性都是随着胡敏酸添加量的增加而逐渐增加;但蛋白酶活性存在差异：松乳菇的蛋白酶活性随着胡敏酸添加量的增加而逐渐增加;变色红菇的蛋白酶活性对胡敏酸不敏感,受其影响不大;铆钉菇的蛋白酶活力在少量的胡敏酸作用下最强,但浓度过高反而抑制其蛋白酶的活性。3）添加碳酸钙后,总体来看,3种菌丝胞外酸性磷酸酶和蛋白酶活性都是添加少量碳酸钙时酶活性最强,随着浓度的增加(如0.1 g),其酶活性开始受到抑制。综上所述,真菌菌丝能够分泌酸性磷酸酶和蛋白酶,这可能是因为这些外生菌根真菌能够促进植物养分吸收和快速生长的原因;有机碳和无机碳的加入可以直接影响真菌菌丝胞外酶的分泌,进而影响土壤内有机磷和有机氮化合物的分解,显示其在土壤碳循环中的作用。     

酪氨酸蛋白磷酸酶(PTPase)参与水杨酸调控蚕豆气孔运动的信号转导

研究酪氨酸蛋白磷酸酶(PTPase)的抑制剂氧化苯胂(PAO)、钒酸钠(NaVO3)和Zn2 对水杨酸(SA)调控蚕豆气孔运动影响的结果表明,0~1mmol·L-1 PAO、0~4mmol·L-1 NaVO3和0~4mmol·L-1Zn2 对光诱导蚕豆气孔开度变化的影响不大,但都可以抑制黑暗或SA诱导的气孔关闭,据此推测,PTPase可能参与SA诱导气孔关闭的信号转导过程。     

Rat Brain Protein Phosphatase 2A: An Enzyme that May Regulate Autophosphorylated Protein Kinases

Abstract: Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B' subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves ( K _i = 0.05 n M ) nearly identical to that reported for skeletal muscle PP2A. The isolated 38-kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross-reactivity with a specific monoclonal antibody (PY-20). Amino acid compositions and sequences of peptides isolated from the 65- and 38-kDa species correspond to regions of the cDNA-deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca²⁺-dependent K⁺-depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45-s time course. The decrease was blocked by 1 n M okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.     

Cloning,Expression, and Characterization of a Thermostable PAP2L2, a New Member of the Type-2 Phosphatidic Acid Phosphatase Family from Geobacillus toebii T-85

Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding a PAP2-like protein, Gtb PAP2L2, which contains 212 amino acids and shows a limited homology to other known PAP2s, especially at conserved phosphatase motifs, and a similar six-transmembrane topology structure. This enzyme was expressed, and purified in Escherichia coli. Recombinant Gtb PAP2L2s from the membrane fractions were solublized with 0.3% (v/v) Triton X-100 and purified by Ni²⁺ affinity chromatography. The purified enzyme showed broad substrate specificity to phosphatidic acid, diacylglycerol pyrophosphate, and lysophosphatidic, but preferred phosphatidic acid and diacylglycerol pyrophosphate in vitro. Gtb PAP2L2 is a thermal stable enzyme with a half-life of 30 min at 60 °C. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Zn²⁺, whereas it was independent of the Mg²⁺ ion, and insensitive to N-ethylmaleimide. The purified recombinant Gtb PAP2L2 was catalytically active and highly stable, making it ideal as a candidate on which to base further PAP2 structure/function studies.     

Acid Phosphatase Complex from the Freshwater Snail Viviparus viviparus L. under Standard Conditions and Intoxication by Cadmium Ions

Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water.     

Immunolocalization of an Acid Phosphatase from Pearl Oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>) and Its In Vitro Effects on Calcium Carbonate Crystal Formation

Distribution of an acid phosphatase, AcPase I, from pearl oyster (Pinctada fucata) in different tissues was investigated via enzyme activity determination and immunohistochemistry. Positive reactions were observed in sections of digestive gland, base of gill filaments, and epithelia of the outer side of the middle fold and the inner side of the outer fold, which indicated AcPase I might participate in processes besides immune defense, such as calcium metabolism or shell formation. Its effects on CaCO₃ crystal formation were studied in vitro. Results revealed that AcPase I inhibited CaCO₃ precipitation in a dose-dependent manner and had no affinity for calcium. CaCO₃ crystals induced by AcPase I exhibited a cluster needle-like morphology, which proved to be aragonite. The morphology and size of the aragonites varied with different concentrations of AcPase I. Our observations described here may provide important clues to further understanding of the correlations between mineralization and immune defense in the oyster.     

Crystal structure of a multi-domain immunophilin from Arabidopsis thaliana: a paradigm for regulation of plant ABC transporters

FKBP42 is a membrane-anchored immunophilin playing a critical role in morphogenesis and development of higher plants. We present the X-ray structure of the cytoplasmic portion of FKBP42 comprising both the FKBP-like domain and the TPR domain at 2.85 A resolution. The data shed light on the probable binding modes of key interaction partners, including HSP90 and two classes of ABC transporters. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.     

Phospholipids: “Greasing the wheels” of humoral immunity

Phospholipids are major structural components of all cellular membranes. In addition, certain phospholipids execute regulatory activities that affect cell behavior, function and fate in critically important physiological settings. The influence of phospholipids is especially obvious in the adaptive immune system, where these macromolecules mediate both intrinsic and extrinsic effects on B and T lymphocytes. This review article highlights the action of lysophospholipid sphingosine-1-phosphate as a lymphocyte chemoattractant, the function of phosphatidylinositol phosphates as signaling conduits in lymphocytes and the role of phospholipids as raw materials for membrane assembly and organelle biogenesis in activated B lymphocytes. Special emphasis is placed on the means by which these three processes push humoral immune responses forward. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.