Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Preferential alkaline phosphatase isoenzyme induction by sodium butyrate

SW-620, a continuous cell line derived from a poorly differentiated human colon carcinoma, produces two alkaline phosphatases. Under basal conditions the heat-stable, term-placental is the major isoenzyme and the heat-labile, liver/bone/kidney form represents a minor component. Exposing SW-620 cells to sodium butyrate causes induction of increased levels of activity accompanied by a striking shift in isoenzyme distribution not observed heretofore. The activity increase is accounted for entirely by augmentation of the liver/bone/kidney isoenzyme, with the term-placental form not being affected. Two other known alkaline phosphatase inducers, prednisolone and hyperosmolality, do not influence specific activity and isoenzyme distribution. The preferential induction of the liver/bone/kidney form of alkaline phosphatase in SW-620 cells may reflect a butyrate-elicited expression of a more differentiated state.     

Analysis of isoenzyme patterns of acid phosphatase in acute leukemias

The status of acid phosphatase isoenzymes was evaluated in cells of patients with acute lymphocytic leukemias or lymphomas by analytical isoelectric focusing in polyacrylamide gels (IEF) on horizontal thin-layer slabs. The isoenzyme patterns were correlated with routine immunological cell surface markers and the relationship of enzyme activity to specific immunological subclasses of ALL is discussed. By isoelectric focusing up to five isoenzyme groups (I-V) containing several isoenzyme were observed. No leukemia specific or additional isoenzyme could be demonstrated. This biochemical characterization showed a marked heterogeneity within two major immunologic subgroups indicating that various differentiation stages of cell maturation could be involved in cALL and T-ALL. According to their degree of maturation along T-cell differentiation axis the leukemic cells displayed no enzyme activity, weak isoenzyme bands or the incomplete or complete isoenzyme pattern seen with normal lymphocytes from human tonsils which were used as controls. The investigation of specific enzymatic patterns can lead to a further definition of subsets of acute leukemias and give insight into lymphopoietic differentiation.     

A homogeneous isoenzyme of human liver acid phosphatase.

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K_i is 4.3 × 10^?7m at pH 4.8 in 50 mm sodium acetate/100 mm sodium chloride. K_i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K_m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K_i for l-(+)-tartrate. The pH dependence of both K_m and V indicate the effect of substrate ionization (pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.     

Role of an acid phosphatase isoenzyme in callus tissue during cytodifferentiation

Summary Both histological and isoenzyme patterns of acid phosphatase were observed in callus tissue of Vigna unquiculate (L.) Walp. up to the tenth passage from its initiation. It was observed that when un-differentiated cells begin to transform into a differentiated condition in the form of tracheids, and xylem vessels, a new acid phosphatase band appears at the anionic end of the polyacrylamide gel. The transformation of living cells into a dead, empty tracheid during cellular differentiation and the biosynthesis of the acid phosphatase enzyme are functionally related to the autolysis of the cell contents and lignin synthesis.     

Elevated total and isoenzyme forms of acid phosphatase in falciparum malaria

The activities of total serum acid phosphatase (E.C. 3.1.3.2) and of two of its isoenzymes, tartrate-resistant acid phosphatase and erythrocyte-specific acid phosphatase were measured in 109 adult male and female patients presenting acute falciparum malaria infection, and a normal, healthy control group comprised of 82 subjects. All the three forms of acid phosphatase were found to be significantly (p<0.05) higher during infection as compared to their activity in the control group. This result suggests that the measurement of acid phosphatase, particularly the erythrocyte isoenzyme, in serum could be potentially used as a biomarker of acute falciparum malaria infection.     

Characterisation of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis

To identify the prevalent Leishmania species in Bangladesh, a total of nine patients aged 4-35 years, were studied; six (66.7%) of them were below 20 years of age. All the patients were clinically diagnosed to have visceral leishmaniasis; their haematological profile was in accordance with leishmaniasis and all were improved after treatment with sodium stibogluconate. All the aspirated materials (eight bone marrows and one splenic aspirate) yielded growth of Leishmania parasite in NNN media; Leishman-Donovan bodies were found in seven (77.8%) of them in a Giemsa stained smear. Aldehyde test (AT) was positive in all the nine cases examined, whereas, complement fixation test (CFT) was positive in seven (77.8%) and indirect fluorescent antibody test (IFAT) in eight (88.9%) cases. In this study, five of the nine isolates from kala-azar patients were characterised by isoenzyme analysis comparing with five WHO reference strains viz., Leishmania (Leishmania) donovani (DD8), L.(L.) donovani (HU3), L.(L.) infantum (IPT-1), L.(L.) tropica (K-27) and L.(L.) major (5-ASKH) using cellulose acetate electrophoresis. By analysing 11 soluble isoenzymes it was found that all five WHO reference strains had distinct electrophoretic mobility of the isoenzymes studied. No interspecies difference was observed amongst the five isolates from kala-azar patients examined and their isoenzyme profiles were consistent with WHO reference strain of L.(L.) donovani (DD8) but different from L.(L.) donovani (HU3).     

Histochemical mapping of certain enzymes in few fresh water teleosts. I. Acid phosphatase.

A detailed investigation of the distribution pattern of acid phosphatase in the different parts of alimentary canal and associated glands of Colisa fasciatus, Macrognathus aculeatus, Notopterus notopterus and Nandus nandus has been made. Though this enzyme shows its hydrolytic activity in all the parts of the digestive system yet its intense activity has been noted in the intestine, pyloric caeca, liver and pancreas of all the 4 fishes. Mucosal and submucosal layers of all the parts of the alimentary canal are the main seat of localization of this enzyme.     

大鼠胆汁淤积型肝病碱性磷酸酶及其同工酶的测定

建立胆汁淤积型肝病大鼠动脉模型,用麦胚凝集素（WGA）亲和色谱法分离总碱性磷酸酶（TALP）中的肝型碱性磷酸酶（LALP）和骨型碱性磷酸酶（BALP）,然后用高效液相色谱法（HPLC）定量;结果显示胆汁淤积型肝病组大鼠TALP于结扎后第5天升高,其中LALP明显升高,且出现较早,BALP基因无变化。本研究检测了大鼠胆汁淤积型肝病过程中血清TALP及LALP的变化情况,探讨LALP作为胆汁淤积型肝病早期辅助诊断灵敏指标的重要意义。     

Quinone-Amino Acid Conjugates Targeting Leishmania Amino Acid Transporters

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.     

Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.     

On the acid phosphatase isoenzymes existing in American Leishmania promastigotes

1. Two partially purified acid phosphatase activities present in American Leishmania promastigote homogenates were characterized by biochemical methods. 2. One isoenzyme acts preferentially on p-nitrophenyl phosphate, is strongly inhibited by 30 mM alloxan, citrate, maleate, malonate and succinate, and strongly stimulated by 3 mM spermine. Its pI is 4.8. 3. The other isoenzyme acts preferentially on beta-glycerophosphate and is resistant to 30 mM alloxan, citrate, maleate, malonate and succinate and also to 3 mM spermine. Its pI is 5.7. 4. Both acid phosphatase isoenzymes have an optimum pH of 5.2, are tartrate-sensitive and strongly membrane-bound, as shown by differential centrifugation and density gradient equilibration. 5. Both isoenzymes were separated by using homogenates prepared in 2% Triton X-100, differential centrifugation, Sepharose 4B/CL-4B gel filtration, ion exchange chromatography and electrofocusing. After this procedure, they were still contaminated with several different proteins. 6. Purification was around 150-fold, with a 32% yield. 7. When these acid phosphatase activities were measured in total homogenates from 12 different Leishmania isolates, p-nitrophenyl phosphatase specific activity values were quite close; beta-glycerophosphatase-specific activity had around a 2-fold variation. 8. This variation was independent from taxonomic classification or infectivity of susceptible hosts.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Acid phosphatase in the guinea-pig oocytes

In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules. Ultracytochemically the reaction product is located in lysosomes and is some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogeneous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.     

Acid phosphatase in the guinea-pig oocytes

Summary In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules, Ultracytochemically the reaction product is located in lysosomes and in some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogenous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Acid phosphatase in Enteromorpha

Extracts of the alga Enteromorpha linza hydrolysed glucose-6-phosphate, p-nitrophenylphosphate 2′-, 3′-, and 5′-adenosine monophosphates with an optimum at pH 5. Cytidine and uridine-5′-nucleoside diphosphates, and 2′-, and 3′-adenosine monophosphates were relatively poorly hydrolysed. Zn²⁺ (10 mM) inhibited the hydrolysis of all substrates, whereas Mg²⁺ (10 mM) may be stimulatory. It is suggested that the hydrolysis of these phosphomonoesters was due to the activity of a non-specific acid phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.2).