Kinetics, Regulation and Mechanisms of Biochemical Reactions

Chock, P B.

Abstract

EPR spectroscopy and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. The reaction of methyglyoxal and L-alanine was used as a model to study the mechanism of advanced glycation end-products formation. The results revealed that three types of free radicals were generated. They are: methyglyoxal dialkylimine radical cation, methylglyoxal radical anion and superoxide anion radical, formed only in the presence of oxygen. The study on the Cu,Zn-SOD mutant, SOD(G93A), reveals a gain-of-function, most likely by the enhancement of the free radical generating activity of the mutant, may involve developing ALS symptoms. Cytosolic Ca(II) oscillations were studied using HeLa cells. The results showed that Ca(II) oscillations are mediated by reversible phosphorylation cycles, which involve the phosphorylation of the IP3 receptor and the Ca(II) pump by CaMKII and the dephosphorylation by calyculin A inhibitable phosphatase(s). The frequency is regulated, directly or indirectly by Ins(1,3,4,5)P4, generated by IP3K, a Ca(II)- calmodulin regulated kinase. We showed that protein ubiquitinylation can be regulated by reversible phosphorylation. This involves the phosphorylation of ubiquitin activating enzyme, E1, and a number of ubiquitin carrier enzymes, E2s, by various kinases, including a novel protein kinase which phosphorylated E2-20kDa. Histochemical analysis showed that lung contains the highest activity of this kinase. Nitric oxide reacts with superoxide anion to form peroxynitrite under oxidative stress. Peroxynitrite is shown to nitrate tyrosine in protein and the nitrated tyrosine cannot serve as substrate for tyrosine kinase, such as lck kinase p56lck, a src-tyrosine kinase. These results revealed that nitration of tyrosine will seriously compromise the regulatory function of tyrosine phosphorylation. Kinetics of fluorescence indicators by electroporated cells reveals that the resealing process is accelerated by Ca(II) or a series of non-ionic synthetic surfactants in low salt medium. This effect is not as pronounced in high salt medium since resealing under this condition is fast.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Heart, Lung, and Blood Institute (NHLBI)
• Type: Intramural Research (Z01)
• Project #: 1Z01HL000202-24
• Application #: 5203459
• Support Year: 24
• Fiscal Year: 1995

Institution

• Name: National Heart, Lung, and Blood Institute
• Country: United States

Publications

Seo, Jae Ho; Lim, Jung Chae; Lee, Duck-Yeon et al. (2009) Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin: Nalpha-terminal acetylation of human peroxiredoxin II. J Biol Chem 284:13455-65

Tanaka, Mikiei; Chock, P Boon; Stadtman, Earl R (2007) Oxidized messenger RNA induces translation errors. Proc Natl Acad Sci U S A 104:66-71

Miyoshi, Noriyuki; Oubrahim, Hammou; Chock, P Boon et al. (2006) Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis. Proc Natl Acad Sci U S A 103:1727-31

Li, Tianwei; Santockyte, Rasa; Shen, Rong-Fong et al. (2006) Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways. J Biol Chem 281:36221-7

Li, Tianwei; Santockyte, Rasa; Shen, Rong-Fong et al. (2006) A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers. Arch Biochem Biophys 453:70-4

Shelton, Melissa D; Chock, P Boon; Mieyal, John J (2005) Glutaredoxin: role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid Redox Signal 7:348-66

Khan, Mohammed A S; Chock, P Boon; Stadtman, Earl R (2005) Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102:17326-31

Chiueh, Chuang C; Andoh, Tsugunobu; Chock, P Boon (2005) Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin. Toxicol Appl Pharmacol 207:96-102

Oubrahim, Hammou; Wang, Jun; Stadtman, Earl R et al. (2005) Molecular cloning and characterization of murine caspase-12 gene promoter. Proc Natl Acad Sci U S A 102:2322-7

Tekle, Ephrem; Oubrahim, Hammou; Dzekunov, Sergey M et al. (2005) Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses. Biophys J 89:274-84