Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases

Antiviral -P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and -phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by -P-borano substituted NDP (NDPB) stereoisomers were also investigated. The Sp-ADPB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPBs. For CK, the Sp-ADPB isomer was a competitive inhibitor; for PK, the Rp-ADPB isomer was a poor competitive inhibitor and the Sp-ADPB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.



ARTICLE

Creatine Kinase–Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis

Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous creatine kinase (CK-B), in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

Comparison of MB Fraction of Creatine Kinase Mass and Troponin I Serum Levels With Necropsy Findings in Acute Myocardial Infarction

Serum levels of troponin and heart-related fraction of creatine kinase (CK-MB) mass are used as diagnostic and prognostic criteria in myocardial infarction , but the relation between those levels and the necropsy-determined size of necrosis has not been tested in human beings.

In this retrospective study, 1-cm-thick transverse sections of the ventricles were cut from the base to the apex in the necropsy hearts of 27 patients aged 47 to 86 years (mean 66, median 69; 19 men). Total and necrotic areas were measured using a computer-linked image analysis system. The weights of the necrotic areas were also calculated.

The correlations of the areas and weights of necrotic myocardium with the highest serum values of CK-MB mass and troponin I, which had been quantified during life by chemiluminescence immunoassays, were verified by Pearson's test; results were considered significant at p <=0.05.

Significant correlations were detected between Creatine Kinase isoenzyme mb (CK-MB) mass peak and infarct size (r = 0.63, p <0.01) and weight (r = 0.69, p <0.01) and between CK-MB mass and highest troponin level (r = 0.73, p <0.01); however, the correlations between highest troponin level and myocardial infarct size (r = 0.31, p = 0.11) and weight (r = 0.35, p = 0.07) were small and nonsignificant.

In conclusion, despite the well-established role of serum levels of troponin as a diagnostic tool for myocardial infarction, their highest values showed poor correlations with the extent of infarct. In contrast, the highest serum level of CK-MB mass was well correlated with myocardial infarct size.


Costa TN, Cassaro Strunz CM, Nicolau JC, Gutierrez PS.
Heart Institute (InCor), Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
Am J Cardiol. 2008 Feb 1;101(3):311-4.