Multiple forms of acid phosphatase activity in Gaucher's disease.

Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to those of the major acid phosphatase activity observed to be present in serum of patients with Gaucher's disease. These data indicate that simple spleen spillage cannot account for the increased levels of serum acid phosphatase in patients with Gaucher's disease.     

Characterization of separated prostatic acid phosphatase forms in normal and castrated rats

We have investigated the effects of castration and androgen replacement on the kinetic characteristics of rat prostatic acid phosphatases (AP). Chromatography on DEAE-cellulose DE-23 and on Sephacryl S-200 allowed the separation of lysosomal (S-1) and secretory (S-3) forms of AP. In addition, these techniques revealed a third enzymic form (S-2), which eluted in the void volume of the Sephacryl S-200 column and which occurred in significant amounts only 15 days after castration. The S-2 form was extremely resistant (K₁ 1500 μM) to L-tartrate inhibition in the 15-day post-castration rat which is in contrast to the behaviour of S-2 (K_i 145 μM) in control animals. The appearance of this unidentified AP form can explain the dramatic drop in the per cent inhibition of total rat AP observed between 7 and 15 days post-castration.     

Consumption of purple sweet potato leaves modulates human immune response： T-lymphocyte functions, lyric activity of natural killer cell and antibody production

AIM: To study the immunological effects of physiological doses of purple sweet potato leaves(PSPL). METHODS: The randomized crossover study(two periods, each lasting for 2 wk) involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in(wk 1) PSPL diet(daily consumption of 200 g PSPL) or a control diet(low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL)(wk 2-3), washout diet(wk 4), and switched diet(wk 5-6). Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines. Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer(NK) cells were determined. RESULTS: The plasma β-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups. Although plasma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells(PBMC) and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary IgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL. CONCLUSION: Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.     

Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

The purple acid phosphatase from beef spleen, which contains two iron atoms per molecule, is EPR silent in its native (oxidized) purple form. Treatment with mild reducing agents results in conversion to a pink, enzymatically active form, which exhibits an unusual EPR signal centered at g approximately equal to 1.77; double integration of the EPR spectrum gives one spin per two iron atoms. A similar EPR spectrum is observed for enzyme reduced anaerobically by one electron, using sodium dithionite. Variable-temperature magnetic susceptibility measurements show that the oxidized and reduced proteins are both antiferromagnetically coupled systems, with S = 0 and 1/2 ground states, respectively. Replacement of one of the iron atoms by zinc produces an FeZn enzyme with full catalytic activity. The FeZn enzyme exhibits a highly temperature dependent g = 4.3 EPR signal, and magnetic susceptibility data are consistent with an S = 5/2 paramagnet. Treatment of the FeZn enzyme with phosphate, a competitive inhibitor, results in sharpening of the EPR spectrum; double integration at 77 K gives one spin per iron. These results strongly suggest the presence of a spin-coupled bimetallic unit at the active site of the enzyme.     

An IgA high-titer cold agglutinin with an unusual blood group specificity within the Pr complex

Abstract A high titer cold agglutinin was present in the serum of a patient with an IgA monoclonal protein. Cold agglutinin activity was confined to the IgA fraction of the separated proteins. The IgA cold antibody appears to have specificity within the Pr complex in that it reacts with adult I, adult i and cord i red cells equally strongly but none of these cells react when treated with papain and neuraminidase. Weak reactions were obtained with dog and rat cells, but only if the cells were not treated with papain or neuraminidase. Monkey, sheep, mouse, hamster, rabbit and guinea pig cells did not react. This pattern of reactions is different from any so far described and may indicate either a qualitative difference, suggesting a further antigen in the Pr complex, or a quantitive difference, suggesting that the Pr₁ antigen is present in a weak form on the cells of some animals such as the rat and dog.     

Properties of an acid phosphatase in pulmonary surfactant.

Lung surfactant, a lipid-protein complex purified from dog lungs, contains a highly active phosphomonoesterase associated with it. This phosphatase is quite specific for the hydrolysis of phosphatidic acid and 1-acyl-2-lysophosphatidic acid. The enzyme possesses many of the characteristics of the microsomal enzyme, phosphatidate phosphohydrolase (EC 3.1.3.4). In addition, we have shown that this enzyme will also convert phosphatidylglycerol phosphate [1-(3-sn-phosphatidyl)-sn-glycerol-1-P] to phosphatidylglycerol [1-(3-sn-phosphatidyl)-sn-glycerol] and Pi. The phosphatidylglycerol phosphate was made available to the surfactant enzyme in a coupled assay by hydrolysis of cardiolipin [1-(3-sn-phosphatidyl)-3-(3-sn-phosphatidyl)-sn-glycerol] by stereospecific cleavage with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus. This enzyme has been previously shown to generate the naturally occurring isomer of phosphatidylglycerol phosphate because it has specificity for the 3-(3-sn-phosphatidyl) group of cardiolipin. Other properties of the surfactant enzyme are discussed in relation to its presence in lung surface active material.     

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69-196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.     

Brucellar pericarditis: 2 different forms of presentation for an unusual etiology]

Two cases of Brucella melitensis pericarditis are reported. Pericardial involvement was the first and almost only manifestation of brucellosis in the first patient while in the second, a significant pericardial effusion was discovered on a routine echocardiogram performed in a patient with clinically florid brucellosis. Some etiopathogenic aspects of this uncommon etiology are discussed.     

Development of an immunoassay for human serum osteoclastic tartrate-resistant acid phosphatase

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget's disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.     

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.     

Bacterial glutamate racemase has high sequence similarity with myoglobins and forms an equimolar inactive complex with hemin.

Glutamate racemase (EC 5.1.1.3), an enzyme of microbial origin, shows significant sequence homology with mammalian myoglobins, in particular in the regions corresponding to the E and F helices, which constitute the heme binding pocket of myoglobins. Glutamate racemase binds tightly an equimolar amount of hemin, leading to loss of racemase activity. Although this enzyme shows homology with aspartate racemase, the latter does not bind hemin. The glutamate racemase gene of Pediococcus pentosaceus has a 795-nt open reading frame and encodes 265-amino acid residues, which form a monomeric protein (M(r) 29,000). Neither racemase has cofactors, but they contain essential cysteine residues [Yohda, M., Okada, H. & Kumagai, H. (1991) Biochim. Biophys. Acta 1089, 234-240].     

Carcinoma of the Bellini duct with an unusual clinical picture

A 37-year-old male patient presented with a Bellini duct carcinoma, at first as a metastatic illness of the paraaortal lymph nodes, without significant radiologic signs of a kidney tumor. Cytological diagnostics did not recognize this tumor. Macroscopically and microscopically the tumor fulfilled the major and minor criteria of Sringly et al., but immunohistochemical findings did not show cell affinity for UEA-1, which, according to the literature, typically confirms its origin from Bellini ducts. This rare neoplasm, primarily found in a younger population, still represents a diagnostic and therapeutical challenge as a result of its aggressive clinical course. In our patient, as in the majority of cases presented in the literature, tumor prognosis was very poor, in spite of aggressive surgical, radium and immune therapy.