Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

The importance of understanding the protein folding pathway and intermediates is well recognized on the basis of extensive studies of protein folding in vitro and in vivo. Creatine kinase (CK) is a typical model for studying unfolding and refolding of proteins due to several interesting properties. Recent studies on the folding of CK show that its partially folded monomeric intermediate is present kinetically and is stable at equilibrium. The present paper contains 33 references as a mini review to characterize the properties of CK from studies on the CK folding pathway. Characterization of these intermediates is an essential step toward understanding the mechanism of protein folding. Some well determined schemes are suggested as protein folding models.

Activation of Calf Intestinal Alkaline Phosphatase by Trifluoroethanol

Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (060%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α helix content. We conclude that the activation of CIAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of CIAP induced by TFE results in the enhancement of CIAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of CIAP can be attained with various secondary structures.

Effects of Glycerol in the Refolding and Unfolding of Creatine Kinase

The effects of glycerol in the refolding, reactivation, unfolding, and inactivation of guanidine denatured creatine kinase were studied by observing the fluorescence emission spectra and the circular dichroism spectra, and by recovery and inactivation of enzymatic activity and aggregation. The results show that low concentrations of glycerol (<25%) improve the refolding yields of creatine kinase, but high glycerol concentrations decrease its recovery. Glycerol favors the secondary structural formation and inhibits aggregation of creatine kinase as proline does. These systematic observations further support the suggestion that low concentrations of glycerol possibly play a chaperone role in the refolding of creatine kinase. In addition, glycerol reduces the inactivation and unfolding rate of creatine kinase, increases the change in transition free energy of unfolding (ΔΔG u) and stabilizes its active conformation relative to the partially unfolded state with no glycerol. In the presence of glycerol, the inactivation and unfolding dynamics of creatine kinase are related to glycerol concentrations. Glycerol blocks the exposure of hydrophobic areas and the dissociation of dimers, and protects creatine kinase against guanidine denaturation in a concentration dependent manner. This study suggests that glycerol as an energy substrate for metabolism and organic components in vivo, assists correct protein folding, maintains adequate rates of enzymatic catalysis and stabilizes the protein secondary and tertiary conformations.

Extraction and DNA Digestion of 5'-Phosphodiesterase from Malt Root

This study investigated the extraction of 5＇-phosphodiesterase from malt root and the degradation of nucleic acids by this enzyme. The extraction used grade precipitation with ammonium sulfate and enzymatic hydrolysis. Samples were assayed using the modified Bradford method and high performance liquid chromatography. The results show that 5＇-phosphodiesterase is isolated by grade precipitation with 30% and 80% saturation of ammonium sulfate and can be utilized to degrade deoxyribonucleic acid. The hydrolysate has four kinds of deoxynucleotides： 5＇-dCMP, 5＇-dTMP, 5＇-dAMP, and 5＇-dGMP. The optimum reaction temperature is 70℃, and the optimum pH is 5.5-6.0 for the reaction. The percentage of deoxynucleotides indicated by the China Pharmacopoeia （2000 edition） in the product is over 70%. The extraction of 5＇-phosphodiesterase from malt root is shown to be possible and economical. Products from the enzYmatic hydrolysate of DNA meet the pharmacopoeia.