Capture of monomeric refolding intermediate of human muscle creatine kinase.
ABSTRACT Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.
Article: Brefeldin A differently affects basal and prolactin-stimulated milk protein secretion in lactating rabbit mammary epithelial cells.[show abstract] [hide abstract]
ABSTRACT: When lactating mammary epithelial cells were treated with prolactin in vitro, numerous small vesicles rapidly accumulated in the Golgi area, and secretion of milk proteins increased. The effects of brefeldin A on these intracellular events were investigated. As observed by electron microscopy, stacks of the median Golgi were not altered after incubation in the presence of 50 nM brefeldin A but were dissociated when the drug concentration was > or = 500 nM. Small vesicles did not accumulate in the Golgi area when mammary cells were incubated in medium containing both prolactin and brefeldin A, whatever the concentration of the latter. Immunofluorescence experiments showed that 50 nM brefeldin A did not modify the localization of the CTR 433 median Golgi protein, but it induced redistribution of trans-Golgi network-associated proteins such as TGN38, AP-1 adaptor and clathrin. These effects occurred in the presence of brefeldin A plus prolactin. Pulse-chase experiments showed that brefeldin A concentrations > or = 100 nM induced the intracellular accumulation of milk proteins, provoked the appearance of immature forms of caseins, and inhibited milk protein secretion. In contrast, concentrations of brefeldin A of < or = 50 nM did not affect basal casein secretion but inhibited the secretagogue effect of prolactin. These data show not only that several biochemical events in the transport of milk proteins which are sensitive to different brefeldin A concentrations occur in lactating mammary epithelial cells, but also that it is possible to inhibit a hormonal stimulus in a selective manner, while the machinery responsible for basal secretion is still active.European Journal of Cell Biology 05/1997; 72(4):324-36. · 2.81 Impact Factor
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ABSTRACT: Eukaryotic proteins expressed in Escherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.Molecular Biotechnology 09/1996; 6(1):53-64. · 2.17 Impact Factor