Reflections about the functional potential of legume proteins A Review

The term “functional potential” was introduced to better approach to the understanding of the relationships between the structure and the functional properties of food proteins. Although in practice the complex of structural features of a protein contributes to its functionality, it is very useful to regard the functional potential of the protein surface on the one side and to look on special effects of protein conformation (role of compact or unfolded state) on the functionality on the other side. This point of view may help to design the best strategy of modification of the protein structure and functionality. The special situation of the oligomeric legume storage protein, i. e. legumin‐like 11 S globulins and vicilin‐like 7 S globulin, and the structural and functional modification of 11 S globulin by limited tryptic hydrolysis and by acylation are discussed in this paper.     

Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex

Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 microM for the formation of a 1:1 REP-Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP-Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an essential activator of the reaction.     

Ursodiol for primary sclerosing cholangitis. Mayo Primary Sclerosing Cholangitis-Ursodeoxycholic Acid Study Group

BACKGROUND: There is no satisfactory medical therapy for patients with primary sclerosing cholangitis. Ursodiol (ursodeoxycholic acid) benefits patients with primary biliary cirrhosis, another cholestatic liver disease. METHODS: From May 1989 to July 1995, we enrolled 105 patients with well-documented primary sclerosing cholangitis in a randomized, double-blind study comparing ursodiol (13 to 15 mg per kilogram of body weight per day in divided doses) with placebo. The primary outcome was the time to treatment failure, defined as death; liver transplantation; histologic progression by two stages (of four) or progression to cirrhosis; the development of varices, ascites, or encephalopathy; sustained quadrupling of the serum bilirubin concentration; marked worsening of fatigue or pruritus; inability to tolerate the drug; or voluntary withdrawal from the study. RESULTS: We analyzed data on the 51 patients in each group with at least 3 months of follow-up; the median follow-up was 2.2 years. There was no significant difference between the groups in time to treatment failure (relative risk of treatment failure in the ursodiol group, 1.01; 95 percent confidence interval, 0.6 to 1.7). During the first two years of follow-up, treatment was unsuccessful in 17 of 32 patients (53 percent) in the placebo group and 16 of 31 (52 percent) in the ursodiol group. There were also no differences in time to treatment failure for patients with early-stage disease or in time to liver transplantation. Ursodiol, but not placebo, was associated with improvement in serum alkaline phosphatase, aspartate aminotransferase, bilirubin, and albumin levels at one and two years. CONCLUSIONS: In a group of patients with well-defined primary sclerosing cholangitis, ursodiol provided no clinical benefit.     

Acetylation of faba bean legumin: conformational changes and aggregation

The effect of progressive acetylation upon the conformation of the 11S globulin legumin from faba bean has been studied using chemical analysis, UV, fluorescence and CD spectroscopy, viscometry and analytical ultracentrifugation. The modification did not induce complete dissociation of the oligomeric protein. Only 30% of the protein was found to be a dissociated 3S subunit after excessive acetylation, whereas 70% was a dimeric legumin aggregate with a molecular mass of about 700 kDa. The aggregation of the highly modified legumin in high‐ionic‐strength buffer solution leads to soluble higher legumin oligomers. The acetylation resulted in a moderate molecular expansion of legumin due to a changed tertiary structure, whereas the far‐UV circular dichroism spectra did not provide definitive evidence of a decrease in domain‐stabilizing β‐sheet conformations in their secondary structure. © 2000 Society of Chemical Industry     

Effects of LHRH-analogues on mitogenic signal transduction in cancer cells

The expression of luteinizing hormone-releasing hormone (LHRH) and its receptors has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Recent studies in our laboratory have demonstrated that the function of LHRH produced by ovarian cancer cells is the inhibition of their proliferation. Dose-dependent antiproliferative effects of LHRH-agonists have been observed by several laboratories in cell lines derived from the above cancers. Interestingly, also LHRH-antagonists have marked antiproliferative activity in most of the ovarian, breast and endometrial cancer cell lines tested so far, indicating that the dichotomy of LHRH-agonists/LHRH-antagonists is not valid for the LHRH-system in cancer cells. In addition, our data suggest that the classical LHRH receptor signal transduction mechanisms known from the pituitary (phospholipase-C, protein kinase C, adenylyl cyclase) are not involved in the mediation of LHRH effects in cancer cells. Data obtained by several groups, including ours, rather suggest that LHRH analogs interfere with the signal transduction of growth-factor receptors and related oncogene products associated with tyrosine-kinase activity. The mechanism of action is probably an LHRH-induced activation of a phosphotyrosine phosphatase, counteracting the effects of receptor associated tyrosine kinase. In our hands, LHRH analogs virtually blocked the EGF-induced MAP-kinase activity of ovarian and endometrial cancer cells. The pharmacological exploitation of this mechanism might provide promising new therapies for these cancers.     

DNA polymerase fidelity: from genetics toward a biochemical understanding

This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful-first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.