The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate

Summary Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.This study was supported by a grant from the Prinses Beatrix Fonds, s'Gravenhage     

Chemiluminescent assay of alkaline phosphatase using dihydroxyacetone phosphate as substrate detected with lucigenin

A sensitive and simple chemiluminescent assay (CL) for alkaline phosphatase (ALP) using dihydroxyacetone phosphate or its ketal (DHAP or DHAP‐ketal) was developed. New substrates were transformed to dihydroxyacetone (DHA) after they were hydrolysed by ALP, which reacts with lucigenin and produces strong chemiluminescence. Under the optimum assay condition, the detection limits were 3.8 × 10^?19 and 1.5 × 10^?18 moles of ALP, respectively. The coefficients of variation (CV) at each points on the standard curve were 0.8–5.4% and 1.8–7.1% (n = 6), respectively. The mechanism of lucigenin CL with DHA was investigated by ESR spectrometry using the spin‐trapping method. The mechanism was speculated as follows: the O₂^? generated by the reaction of DHA and O₂ in alkaline solution reacts with lucigenin, and then emit light. The proposed CL assay was applied to the enzyme immunoassay of 17β‐oestradiol, using ALP as a label enzyme. The measurable range of 17β‐oestradiol was 15–4000 pg/mL, and the proposed method was four times more sensitive than the colorimetric assay for ALP by using 4‐nitrophenyl phosphate as substrate. Copyright © 2002 John Wiley & Sons, Ltd.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.     

ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.     

3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca-ATPase

3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca²⁺-ATPase in the absence of Ca²⁺, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca²⁺, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca²⁺ concentration produces a bell-shaped curve with a maximum at 50 μM Ca²⁺. At optimal Ca²⁺ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca²⁺, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca²⁺ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca²⁺-ATPases.     

Quantification of tartrate resistant acid phosphatase distribution in mouse tibiae using image analysis

Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm² region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p>0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r²=0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.