Catalytic behaviour of acid phosphatase immobilized on natural supports in the presence of manganese or molybdenum

Synthetic complexes were formed by interaction between acid phosphatase and either tannic acid or natural allophanic clay, and used as model systems to simulate enzymatic reactions occurring in heterogeneous environment. The presence of Mn and Mo on the kinetic constants of the immobilized phosphatase was also tested. Complexes were prepared at 30 °C by interaction of acid phosphatase and tannic acid or allophanic clay in the presence and absence of Mn or Mo. The effect of the addition order of the metal (micronutrient in soils) to the clay–enzyme complexes was evaluated, as well. acid phosphatase immobilized on tannic acid and mineral clay showed variable activity levels. When phosphatase was immobilized on tannic acid, a recovery of about 51% of the initial enzymatic activity and a decrease of 17% of its catalytic efficiency was measured. The presence of Mn and Mo decreased the Vmax of phosphatase–tannic complexes as compared with that of the free phosphatase. All the added phosphatase molecules were immobilized onto the clay, when it was used as the immobilizing support. Phosphatase–clay complexes showed an increase of both enzymatic activity (higher Vmax value) and substrate affinity (lower Km values) as compared to the free enzyme, resulting in an increase by about 65% of the catalytic efficiency. The presence of Mn added at the same time with enzyme and clay decreased the enzymatic activity of the immobilized enzyme. However, when Mn was applied after the interaction of the enzyme with the clay, no significant effects on the residual activity of the immobilized enzyme were observed. Conversely, the order of the addition of Mo to the clay–enzyme complexes strongly influenced the activity and the kinetic behaviour of the immobilized enzyme. Moreover, Mo had higher inhibitory effects than Mn on phosphatase immobilized on both supports. The overall results seem to suggest that the immobilization of acid phosphatase on clay not only preserved but also enhanced the activity of the enzyme as compared with organic matter.

ARTICLE

Significance of Serum Tartrate-Resistant Acid Phosphatase in Rheumatoid Arthritis.

Abstract Human serum contains two related isoforms of tartrate-resistant acid phosphatase namely TRACP 5a and TRACP 5b. Serum TRACP 5a protein is increased in about 1/3 of RA sera. This study was undertaken to examine the significance of serum TRACP isoforms 5a and 5b as disease markers of inflammation and bone destruction in rheumatoid arthritis (RA). 118 patients were recruited including 50 with RA (25 with nodules), 26 with osteoarthritis (OA) and 42 with other rheumatic diseases. 26 healthy adults served as controls. Serum TRACP-5a activity, TRACP-5a protein and TRACP-5b activity were determined by in-house immunoassays. C-reactive protein (CRP) was determined by in-house immunoassay using commercial antibodies and CRP. Other commercial markers included bone-specific alkaline phosphatase (bALP), C-telopeptides of type-I collagen (ICTP), cartilage glycoprotein-39 (YKL-40), and IgM rheumatoid factors (IgM-RF). Mean TRACP-5a protein was significantly elevated only in RA compared to healthy control and other disease groups. TRACP 5a protein correlated significantly only with IgM-RF in RA. Among RA patients, mean TRACP 5a protein and IgM RF were significantly higher in nodule formers. In contrast, TRACP-5b activity was slightly elevated in RA and correlated with bALP. ICTP and YKL-40, but not with IgM-RF or CRP. Mean TRACP-5b activity was no different in RA patients with or without nodules. TRACP isoforms could be useful disease markers in RA: TRACP-5a protein may be a measure of systemic inflammatory macrophage burden and disease severity. TRACP-5b activity is a marker for osteoclast number and perhaps local or systemic bone destruction.

SH3BP2 is an Activator of NFAT Activity and Osteoclastogenesis

Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLCγ1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

Steven A. Lietmana, b, Lihong Yina, b and Michael A. Levinec

aDepartment of Orthopaedic Surgery, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

bDepartment of Biomedical Engineering, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA

cThe Division of Endocrinology, The Children’s Hospital of Philadelphia, 34th & Civic Center Boulevard, Philadelphia, PA 19104, USA

ARTICLE

Utility of serum tartrate-resistant acid phosphatase (TRACP5b) as a bone resorption marker in patients with chronic kidney disease: independence from

Background: Serum tartrate-resistant acid phosphatase (TRACP) 5b levels were assessed in predialysis patients with chronic kidney disease (CKD). The aim of the study was to establish the usefulness of a new assay for TRACP5b in assessing bone turnover in these patients.

Methods: Serum concentrations of two bone resorption markers, TRACP5b and N-Terminal cross-linking telopeptide of type I collagen (NTX-I); two bone formation markers, bone specific alkaline phosphatase (Bone ALP) and intact osteocalcin (OC[1-49]); and parathyroid hormone (PTH) were measured in 98 predialysis CKD patients.

Results: Log serum TRACP5b and other bone markers were significantly negatively correlated with glomerular filtration rate (GFR) and positively correlated with log serum PTH, suggesting an increase in serum bone markers with development of secondary hyperparathyroidism.

Multiple regression analysis including age, gender, BMI, the presence of diabetes, GFR, and log serum PTH showed an association of log serum PTH with log serum TRACP5b and other bone markers. GFR was associated with log serum NTX-I and log OC[1-49], but not with log serum TRACP5b or log Bone ALP. These data show that renal dysfunction does not influence serum TRACP5b and Bone ALP, but has an influence on NTX-I and OC[1-49].

Conclusion: Serum TRACP5b may be a good marker for serum bone resorption in predialysis CKD patients, since it is not affected by renal dysfunction.

Yamada S, Inaba M, Kurajoh M, Shidara K, Imanishi Y, Ishimura E, Nishizawa Y.
Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.Clin Endocrinol (Oxf). 2008 Jan 23