epsilon-crystallin from duck eye lens comparison of its quaternary structure and stability with other lactate dehydrogenases and complex formation with alpha-crystallin.

Taxon-specific epsilon-crystallin (epsilonC) from duck eye lens is identical to duck heart muscle lactate dehydrogenase. It forms a dimer of dimers with a dissociation constant of 2.2 x 10-7 M, far beyond the value observed for other vertebrate lactate dehydrogenases. Comparing the characteristics of wild-type epsilon-crystallin with those of three mutants, G115N, G119F and 115N/119F, representing the only significant peripheral sequence variations between duck epsilonC and chicken or pig heart muscle lactate dehydrogenase, no significant conformational differences are detectable. Regarding the catalytic properties, the Michaelis constant of the double mutant 115N/119F for pyruvate is found to be decreased; for wild-type enzyme, the effect is overcompensated by the high expression level of epsilonC in the eye lens. As taken from spectral analysis of the guanidine-induced and temperature-induced denaturation transitions, epsilonC in its dimeric state is relatively unstable, whereas the native tetramer exhibits the high intrinsic stability characteristic of common vertebrate heart and muscle lactate dehydrogenases. The denaturation mechanism of epsilonC is complex and only partially reversible. In the case of thermal unfolding, the predominant side reaction competing with the reconstitution of the native state is the kinetic partitioning between proper folding and aggregation. alpha-Crystallin, the major molecular chaperone in the eye lens, inhibits the aggregation of epsilonC by trapping the misfolded protein.

[Biological activity of E. coli expressed BMP-4]. / Biologische Aktivität von E.-coli-exprimiertem BMP-4.

BMP-4 is physiologically present in low concentrations in human bone matrix. So far the protein has only been produced in small quantities by expression in mammalian cell cultures. In this study we investigated the biological activity of E. coli-expressed BMP-4. In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium. The addition of 0.4 microgram BMP-4 induced cartilage formation in 1/8 samples while 4 micrograms BMP-4 showed chondroneogenesis in 2/10 samples. When the BMP-4 concentration was increased to 40 micrograms, new cartilage formation was seen in 5/7 samples. In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice. Amounts of 10 micrograms rhBMP-4 and more (up to 100 micrograms) constantly induced heterotopic ossicle formation. BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats. While 0.4 microgram BMP-4 showed no bone or cartilage formation, the amount of 40 micrograms BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples. The results prove that E. coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.

[The biological activity of E. coli-expressed BMP-4]. / Biologische Aktivität von E.-coli-exprimiertem BMP-4*

BMP-4 is physiologically present in low concentrations in human bone matrix. So far the protein has only been produced in small quantities by expression in mammalian cell cultures. In this study we investigated the biological activity of E. coli-expressed BMP-4. In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium. The addition of 0.4 µg BMP-4 induced cartilage formation in 1/8 samples while 4 µg BMP-4 showed chondroneogenesis in 2/10 samples. When the BMP-4 concentration was increased to 40 µg, new cartilage formation was seen in 5/7 samples. In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice. Amounts of 10 µg rhBMP-4 and more (up to 100 µg) constantly induced heterotopic ossicle formation. BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats. While 0.4 µg BMP-4 showed no bone or cartilage formation, the amount of 40 µg BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples. The results prove that E. coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.