The Role of Calreticulin Transacetylase in the Activation of Human Platelet Nitrite Reductase by Polyphenolic Acetates

Abstract

Nitric oxide (NO) is known to play an important role in the regulation of physiological functions. Various cell types are capable of synthesizing NO mainly from L-arginine with the participation of nitric oxide synthase (NOS). The action of NOS on L-arginine is the principal pathway to meet the tissue requirements of NO. The additional demand of NO in certain conditions of stress could be met by inducible nitric oxide synthase (iNOS). Several biological phenomena such as hypoxia is beset with the conditions where NOS is compromised, where the utilization of nitrates for the production of NO is encountered. 1) Nitrite reductase (NR) originally described in plants is discernible in microorganisms as well as mammalian cells. Panesar and Chan reported the NR activity in Mouse Leydig Tumor Cells and was attributed to mitochondrial respiratory chain complex III. Also, NO was found to be generated by the utilization of nitrite under basal conditions.2) Nitrate-NR system comprising of protein components such as pyridine nucleotides, flavoproteins and cytochromes are reported to be present in the mitochondria as well as the endoplasmic reticulum for the purpose of NO formation.1) In addition nitrite can function as a signaling molecule independent of the formation of NO.3) The oxidoreductase such as the cytochrome P-450 reductase (CYPR) is known to reduce inorganic nitrate to nitrite and then to NO. The reduction of nitrite to NO is ascribed to various factors including mitochondrial enzymes, polyphenols and protons. Keeping in view of the cardinal role played by NO in the physiology of the organisms it has become necessary to study the formation of nitrite and conversion to NO. It is worth mentioning that nitrate, mimics the role of NO in conditions such as normoxia and ischemic-reperfusion. 4) NO generated by reduction of nitrite by enzymes of cytochrome P-450 family is also responsible for the activation of cyclic guanosine monophosphate (cGMP) signaling pathway. 5,6) The extensive investigations carried out in our laboratory deciphered the identity of TAase with Calreticulin (CRT), a resident protein of the endoplasmic reticulum and consequently the TAase was given a name "Calreticulin transacetylase" (CRTAase). 7) Earlier work from our laboratory highlighted the presence of CRTAase in human platelets and activation of platelets NOS by polyphenolic acetates (PA) catalyzed by CRTAase.8) Purified human placental CRTAase mediated acetylation of neuronal nitric oxide synthax (nNOS) by 7,8-diacetoxy-4-methylcoumarin (DAMC) was demonstrated. 9)Our earlier investigations strongly indicated the CRTAase

Effect of storage on levels of nitric oxide metabolites in platelet preparations

“…The reaction between nitrite and oxyhemoglobin, which generates nitrate and methemoglobin, is possible and this might have contributed to nitrite decrease in PLTs during storage. Although Arora and coworkers 52 reported that cytochrome P‐450 reductase plays a role as a nitrite reductase in human PLTs, they used nonphysiologic concentrations of nitrite (∼100 µmol/L) to observe NO generation and we observed no evidence that higher concentrations of nitrite than those normally present in plasma affected NO production in PLTs. Therefore, there is no substantial evidence so far that human PLTs or WBCs express specific nitrite reductases under physiologic conditions.…”

Comparison of Protein Acetyltransferase Action of CRTAase with the Prototypes of HAT

“…The apparent M.wt. of TAase shifted from 60 kDa to 55 kDa which was found consistent with Ca 2+ dependent electrophoretic mobility shown by other Ca 2+ -binding proteins [20, 56, 57]. The identity of TAase as CR was further confirmed by immunoreactivity with anti-CR antibody.…”

“…This finding characterized polyphenolic acetates (PA) unlike the parent polyphenols, were effective enhancers of intracellular nitric oxide. Further, nitrite reductase was also profoundly activated by DAMC possibly by CRTAase catalyzed acetylation of reductase [56]. These findings implicate the usefulness of DAMC in ameliorating the conditions of hypoxia where the NOS activity is known to be compromised.…”

“…Utilizing this procedure TAase could be conveniently assayed (Figure 1). TAase from the microsomes of rat liver, human placenta, and bovine lung was purified to homogeneity [19, 20, 56]. The N-terminal sequencing of the purified TAase from rat liver and human placenta was performed, and the sequences were found to show 100% identity with CR [19, 20].…”