Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus.

In order to identify groups essential for the activity of endo-beta-N-acetylglucosaminidase H (Endo H), all 8 glutamate residues, all 19 aspartates, and both tryptophans were individually substituted with glutamines, asparagines, and phenylalanines, respectively, by oligonucleotide site-directed mutagenesis. Only variants D170N, D172N, and E174Q were found to have specific activities significantly less than wild-type Endo H. Another variant, D173N, did not produce detectable amounts of protein. Wild-type enzyme was found to have a bell-shaped pH activity profile, which was retained in the essential aspartate mutants, but E174Q lost the basic pH limb of the curve, indicating that E174 is good candidate for the proton donating group necessary for catalysis. The general base needed for activity could not be unambiguously identified; although, of the essential aspartates, D172 is the only one conserved in other related glucosidases.

Extracellular enzymes: gene regulation and structure function relationship studies.

The first conclusion that one could make from the literature covered in this section is that most single mutations in subtilisin BPN'n do not cause major structural alterations. Even multiple mutations, though they may cause local minor perturbations at each of the altered sites, do not affect the overall structure to a large degree. Bott and Ultsch (1986) observed that the subtilisin BPN' structure is very tolerant of single mutations, and this tolerance may have been necessary for survival of the enzyme during the course of evolution. This structural tolerance is not all that surprising if one considers that the structure of subtilisin Carlsberg is very similar to that of subtilisin BPN' while the protein sequences differ by 31%. A superposition of the 274 alpha-carbon atoms of the two enzymes gives a root mean square (rms) deviation of 0.053 nm, a value indicating significant structural similarity (McPhalen and James 1988). Furthermore, the fungal enzyme proteinase K, which is classified as part of the subtilisin family, has approximately 38-40% sequence homology with bacilli subtilisins, particularly in the catalytic site and substrate-binding regions (Betzel et al. 1988). For these sequence-homology regions there is also a structural similarity indicated by a least squares superposition of alpha-carbons giving an rms deviation of 0.11 nm (Betzel et al. 1988). Thermitase, also a member of the subtilisin family, has 47% sequence homology to subtilisin BPN' (Gros et al. 1989). If the best 203 alpha-carbon atoms are superposed, then an rms deviation of 0.05 nm is obtained (Gros et al. 1989). Apparently a significant amount of sequence variation still allows for overall structural similarities in the subtilisin family of enzymes. Though the overall structure of subtilisin is not easily perturbed by single or even multiple mutations, it is clear from the evidence reviewed here that single mutations can lead to very significant effects on the catalytic efficiency, substrate preference, and stability of the enzyme. Analyzing the structural alterations in subtilisin mutants will lead to an understanding of the molecular effects of the mutations at the atomic level. This understanding enables investigators to model and predict the effects of other substitutions, and allows them to focus their efforts on those mutants that are most likely to have the desired properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and sequencing of human cholesteryl ester transfer protein cDNA.

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.

Isolation and specificity of a Mr 74,000 cholesteryl ester transfer protein from human plasma.

A cholesteryl ester transfer protein was isolated from human plasma whose ligand specificity, molecular weight, and amino acid composition are significantly different from those of proteins previously reported to have this activity. The protein, purified about 100,000-fold from plasma, is rich in hydrophobic amino acids, especially leucine. It has a molecular weight of 74,000 and an isoelectric point of 5.2. The protein's transfer rate for cholesteryl ester between each of the major plasma lipoprotein classes is similar, and rapid compared to the transfer of triacyglycerol, regardless of the overall lipid composition of the donor and acceptor lipoprotein. These data suggest that this protein functions primarily in the transfer of cholesteryl esters between plasma lipoproteins.

The epithelial differentiating activity in vivo of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl) -1-propenyl]benzoic acid and 4,4-difluororetinoic acid.

Female rats fed on a vitamin A-deficient diet from weaning were oophorectomized after introitus and used to test analogues of all-trans-retinoic acid for epithelial differentiation activity by the vaginal-smear assay. Several modifications have been made in the assay; housing facilities were modified, the diet changed and the existing scoring system for the assay altered. The arotinoid (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1 -propenyl] benzoic acid was 12-fold more active than all-trans-retinoic acid, which had a 50% effective dose (ED50) of 80 pmol/vagina. The fluorinated analogue 4,4-difluororetinoic acid had an ED50 of 2.5 nmol/vagina and was therefore 30-fold less active than all-trans-retinoic acid.

The growth-supporting activity of a retinoidal benzoic acid derivative and 4,4-difluororetinoic acid.

Two synthetic retinoids were examined for their ability to support growth in male vitamin A-deficient rats. One of the compounds, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 -propenyl]-benzoic acid (TTNPB), was found to be highly effective; it was 35-fold more active than all-trans-retinoic acid. Thus, the in vivo results were in agreement with the in vitro activity of this compound published by previous investigators, and support the view that this compound may be useful in determining the molecular mechanism of action of the retinoids. Another analog, 4,4-difluororetinoic acid, was only 12% as effective as retinoic acid. However, the possible instability of this compound and the electronegativity of the fluoro groups prohibited conclusions concerning the biological function of metabolic modification on the 4 position of retinoic acid.

The biological activity of rat intestinal retinoic acid metabolites in the vaginal smear assay.

Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-3H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [3H]retinoic acid. The biological activity of all discernible metabolites was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-beta-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action.