• Journal of Life Science
• /
• v.8 no.2
• /
• pp.181-188
• /
• 1998

Two distinct ADP-ribosyltransferases, termed Yac-1 and Yac-2 from mouse lymphoma cells were recently cloned and characterized. Yac-1 enzyme possesses ADP-ribosyltransferases activity. In contrast, Yac-2 has significant NAD glycohydrolase activity and may preferentially hydrolyze NAD. Yac-2 possesses a glutamate at position 219 adjacent to the two consdrved glutamic acid residues. To study the effect of Glu-219 on enzyme activities, Glu-219 was mutagenized to Glutamine (E219Q) or alanine (E219A) using a two-step recombinant polymerase chain reaction procedure. Replacing Glu at position 219 with Gln or Ala resulted in 56 (E219Q) or 66% (E219A) reduction in ADP-ribosyltranferase activity. The NAD glycohydrolase activity of Yac-2 protein were not altered by the mutations. These results indicate that Glu-219 in Yac-2 enzyme plays an important role in ADP-ribosyltransferase, but not NAD glycohydrolase activity.

• Journal of Life Science
• /
• v.8 no.5
• /
• pp.486-491
• /
• 1998

Previously, we have cloned and characterized two ADP-ribosyltransferases (Yac-1 and Yac-2) from mouse Iym-phocyte. Yac-2 transferase contains significant NAD glycohydrolase activity as well as ADP-ribosyltransferase acti-vity. Yac-2 has an arginine at position 221 between two conserved glutamic acids. To investigate the significance of Arg-221 on enzyme activities, Arg-221 was mutagenized to Glu (R221E) and to Ala (R221A). Mutants R221E and R221A were active as wild type for ADP-ribosyltransferase and NAD glycohydrolase activity, suggesting that the arginine 221 in Yac-2 does not play a major role in enzyme activities.

• Journal of Life Science
• /
• v.8 no.2
• /
• pp.189-196
• /
• 1998

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various animal cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. A similar notif was observed in the sequence of the mouse lymphocyte ADP-ribosyltransferase (Yac-s). Yac-2 has significant NAD glycohydrolase activity as well as ADP-ribosyltransferase activity. To verify the role of the Glu-rich motif in Yac-2, site-directed mutagenesis was performed. Mutants E220Q, E220A, E222A were inactive for ADP-ribosyltransferase activity. For NAD glycohydrolase activity, E220A, E222Q, and E222A were inactive. In contrast, E220Q was active as wild-type. Thus, Glu-220 and Glu-222 in Yac-2 are critical for ADP-ribosyltransferase and NAD glycohydrolase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the Yac-2 enzyme activity.

• Tuberculosis and Respiratory Diseases
• /
• v.41 no.5
• /
• pp.489-493
• /
• 1994

Background: Nicotinamide adenine dinucleotide glycohydrolase(NADase) is located on the surface of the cells. It is bound by glycosylphosphatidylinositol(GPI)-linkage, which can be cleaved by bacterial PI-specific phospholipase C(PI-PLC). Recently, it was studied that NADase was increased in infected tuberculosis animal, but absolute NADase is uncertainly increased because of high NADase in Mycobacterium tuberculosis. Therefore, we studied pure NADase activity in red blood cells of normal person and patients of tuberculosis. Method: We evaluated the 19 healthy adults and 16 tuberculosis infected patients, and then, the latter cases were evaluated after 3 months antituberculosis therapy. NADase activity was calculated by scintillated counting of cleaved radioactive [carbonyl-$^3H$] nicotinamide Result: NADase activity was $2021.1{\pm}824.0\;pmol/min/10^6$ erythrocytes in healthy adults vs. $3339.0{\pm}1568.0$ in tuberculosis infected patients, and was $3339.0{\pm}1568.0$ in pretreated patients vs. $2238.6{\pm}1013.1$ in same 3 months treated patients. Conclusion: NADase activity of erythrocytes is elevated in tuberculosis infection, and normalized afer antituberculosis therapy. Therefore, we suggested NADase activity as the new diagnostic and therapeutic indicator.

• Archives of Pharmacal Research
• /
• v.15 no.2
• /
• pp.146-151
• /
• 1992

The aim of this study was to evaluate capability of pertussis toxin (PT) to active mouse macrophages. The investigations were undertaken to determine whether the role played by this toxin required the A-protomer of the toxin to ADP-ribosylate a guanine nucleotide binding protein (a class I activity) or was dependent on the binding of B-oligomer of the toxin to the surface of target cells (a Class II activity). The results of these experiments have established that the mechanism of macrophage activation with PT seems to be dependent upon a Class II activity of the toxin.

• Korean Journal of Clinical Pharmacy
• /
• v.8 no.1
• /
• pp.29-34
• /
• 1998

Murine CD38 is a 42 kDa type II glycoprotein expressed on cell surface of both B and T lymphocytes. CD38 is a multifunctional enzyme that catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD and cADPR hydrolase activity of CD38 catalyzes the hydrolysis of cADPR to ADP-ribose (ADPR). And also, CD38 has the catalytic activity of NAD glycohydrolase (NADase) which catalyzes the hydrolysis of catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD to ADPR. In this study, we attempted to purify CD38 from mouse lymphocytes by using the immobilized anti-CD38 monoclonal antibody. The single step immuno-affinity column chromatography resulted in homogeneous purification, showing a single protein of 42 kDa on a SDS polyacrylamide gel. We have investigated the effects of various inhibitors on the enzyme activities of the purified CD38. Cibacron blue (0.5 mM) inhibited all three enzyme activities of CD38, NADase, ADP-ribosyl cyclase and cADPR hydrolase activities. ADPR (2 mM) showed inhibitory effect on both cADPR hydrolase activity and NADase, but not on ADP-ribosyl cyclase activity. However, ATP (2 mM) inhibited only cADPR hydrolase activity. $Zn^{2+}$ (1 mM) showed similar inhibitory effect as that of ADPR, but activated cyclase activity These results suggest that CD38 has three different catalytic activity domains which might be differentially regulated by their specific inhibitors.

• Toxicological Research
• /
• v.17
• /
• pp.329-333
• /
• 2001

Apoptosis is the normal physiological process of cell death essential for the maintenance of homeostasis. The function of nicotinamide adenine dinucleotide (NAD) and adenine diphosphate (ADP) ribosylation (transfer of ADP-ribose to proteins) reactions in modifying apoptosis have recently been of great interest. Recently. CD38. a type 2 transmembrane glycoprotein expressed in hematopoietic and non hematopoietic cell lines. has been reported to possess NAD glycohydrolase activity (Han. 1999) and PC-1 and CD38 NADase regulates T cells by inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycan (Hozada et al., 1999). Sindhuphak et al. (2000) has reported that cervical cancer cells can be differentiated from normal cells by using FTIR (Fourier-Transformed Infrared) technique. which has characterized shifts to be due to the phosphodiester bond in nucleic acid. protein amide I&II. carbohydrate and glycogen bands. Mechanisms how phosphodiester bond shift in cervical cancer cells as compared to control cells remain to be elucidated. Suramana et al. (2000) as well as Lohitnavy and Sinhaseni (1998) have studied methomyl and colchicine effects in rat splenocytes. Lactate Dehydroge-nase Isozymes 3 (LDH3) and LDH4 were observed to increase transiently and subsided in plasma of rats exposed to 6~8 mg/kg methomyl after 48 hours. Phosphodiester bond shift of nucleic acid. detected by FTIR. was also reported (Suramana et al., 2000). We report here, after analysis of bond shift patterns. a similar bond shifts detected by FTIR spectrum observed in human cervical cells and splenocytes of rats exposed orally to 2~8 mg/kg methomyl as well as rats exposed to colchicine 2~6 mg/kg orally.