Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium

Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 +/- 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.0. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.     

Purification of human plasma retinol-binding protein by hydrophobic interaction chromatography

Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported.     

Crystallization of human plasma apo-retinol-binding protein

Crystals of three forms of human plasma apo-retinol-binding protein have been obtained using the procedure described for the holoprotein. The apoprotein was prepared by a novel method, which uses hydrophobic interaction and immobilized dye chromatography. The three forms were separated by fast protein liquid chromatography. All of the crystals are isomorphous and diffract to 2.5 Å resolution. These crystals will be useful for studies of the mechanism of binding of retinol to its carrier using X-ray diffraction techniques.     

New crystalline derivatives of bovine liver rhodanese

Mitochondrial bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase) has been crystallized in the form deprived of the transferable sulfur. The essential condition for crystallization was the removal of oxygen. Crystals of the sulfur-free enzyme are isomorphous with the previously characterized crystals of the sulfur-substituted enzyme. The new crystal species can react with either thiosulfate or selenosulfate to form the catalytic intermediate and, subsequently, with cyanide to form the corresponding product. Furthermore, the enzyme active site can be alkylated by iodoacetic acid.     

Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.     

The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein.

1. The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol-binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three-dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44-61]. 2. Models were developed for the three-dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three-dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350-400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.