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Biomedical subjects

C F Brewer

Publications and source records attributed to C F Brewer.

108 records · Page 6Linked to original sources
PubMed1978

Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

Apoproteins↗
PubMed1978

Structure-activity study of the inhibition of microtubule assembly in vitro by podophyllotoxin and its congeners.

This study investigates the inhibition of microtubule assembly in vitro by podophyllotoxin and its derivatives, which include in part the antitumor compounds 4'-demethylepipodophyllotoxin ethylidene beta-D-glucoside (VP-16-213) and 4'-demethylepipodophyllotoxin thenylidene beta-D-glucoside (VM-26); the cyclic ethers, cyclic sulfides, and cyclic sulfones of podophyllotoxin and deoxypodophyllotoxin; epipodophyllotoxin; picropodophyllotoxin; and several 4'-demethyl compounds. The inhibitory activity of these derivatives is sensitive to the configuration and size of substituents at position 4 in ring C and to steric features of substituents at position 12 in ring D. Decreasing activity correlates with the increasing size of the substituent at position 12, as indexed by their van der Waals radii. These results suggest that rings C and D of these drugs are involved in their interaction with the podophyllotoxin-binding site in tubulin.

Animals↗
PubMed1977

Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by alpha- and beta-glucosidase.

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) D-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-D-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. D-Glucal apparently binds to the enzymes with essentially the same overall orientation as the D-glucosyl moiety of glycosidically linked substrates (with the double bond of D-glucal lying essentially in the plane of the similarly bound D-glucosyl group). Thus, the alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-D-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of D-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That D-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either alph- or beta-anomeric configuration.

Candida↗
PubMed1975

13C NMR studies of the interaction of concanavalin A with saccharides.

The binding of alpha- and beta-methyl-D-glucopyranoside and beta-(o-iodo-pheryl)-D-glucopyranoside to concanavalin A has been studied by carbon-13 nuclear magnetic resonance techniques. The kinetics and binding orientations of these saccharides relative to the transition metal ion site in the protein have been determined.

Binding Sites↗
PubMed1975

Carbon-13 nuclear magnetic resonance study of chondroitin 4-sulfate in the proteoglycan of bovine nasal cartilage.

The Fourier transform 13C nuclear magnetic resonance spectra of bovine nasal cartilage proteoglycan subunit and complex and whole bovine nasal cartilage were obtained and compared with that of chondroitin 4-sulfate. The spectrum of chondroitin 4-sulfate in solution revealed multiple resolvable resonances with linewidths that are consistent with considerable segmental motion in the polysaccharide chain. The spectra of proteoglycan subunit and complex in solution and that of whole cartilage were very similar to that of free chondroitin 4-sulfate chains. This indicates that the linkage of multiple chondroitin sulfate chains to proteoglycan core protein and the association of proteoglycan with collagen and other constituents of cartilage matrix does not significantly alter the structure and motions of these chains.

Animals↗
PubMed1973

Solvent proton magnetic relaxation dispersion in solutions of concanavalin A.

Concanavalin A, a protein isolated from jack beans, exhibits several important biological properties, all of which are related to its ability to bind and precipitate specific polysaccharides. Concanavalin A is a dimer at pH 5.6, and has one transition-metal and one calcium-ion binding site per monomer unit of molecular weight 27,000. Both metal-ion sites must be occupied for the protein to be active. It is of interest to determine the role of the transition metal ion in Concanavalin A and its relationship to the sugar binding activity of the protein. We report the magnetic field and temperature dependences of the spin-lattice magnetic relaxation rates of solvent protons in aqueous solutions of zinc and manganese derivatives of Concanavalin A, and the influence of monosaccharide binding on these rates. The results of a leastsquares fit of the data to the theory, with five adjustable parameters, indicate that there is one rapidly exchanging water molecule ligand on the Mn(2+) ion, with a residence lifetime of 2.5 musec at 25 degrees , and with its protons 0.27 nm (2.7 A) from the Mn(2+) ion. We find that at low magnetic fields (proton Larmor frequencies below about 10 MHz), the correlation time for the dipolar interaction between the Mn(2+) electronic spin moment and the protons on the water ligand is the spin-lattice relaxation time tau(S) of the Mn(2+) moment, but that at higher magnetic fields the correlation time for the dipolar interaction is determined by the Brownian rotational tumbling of the protein, because of the substantial variation of tau(S) with magnetic field. Monosaccharide binding to manganese Concanavalin A has little effect on the relaxation rates of solvent protons, a result that indicates that the sugars do not bind directly to the transition metal in the protein.

Concanavalin A↗
PubMed1973

Binding of 13 C-enriched -methyl-D-glucopyranoside to concanavalin A as studied by carbon magnetic resonance.

Binding of alpha-methyl-D-glucopyranoside, uniformly enriched with 14% (13)C, to zinc and manganese derivatives of concanavalin A at pH 5.6 was studied by pulsed Fourier transform carbon magnetic resonance techniques. Spin-lattice relaxation (T(1)) of the [(13)C]carbons of the sugar was measured in the absence and presence of the two transition metal derivatives of the protein. In the presence of the manganese derivative of concanavalin A, selective relaxation of the sugar carbons was observed. The values for T(1) reflect different distances between the carbons of the bound sugar and the manganese ion. Calculation of the distance between the manganese ion and each carbon of the sugar permit the 3-dimensional orientation of the bound sugar to be determined relative to the transition metal site in the protein. The results indicate that alpha-methyl-D-glucopyranoside binds to the protein in the Cl chair conformation with its 3- and 4- carbons closest to the manganese ion at a mean distance of 10 A.

Binding Sites↗
PubMedDate not supplied

Substrates for microsomal azoreductase. Hammett substituent effects, NMR studies, and response to inhibitors.

In previous studies on azoreduction by microsomal cytochrome P-450, we identified two classes of substrates structurally related to 4-dimethylaminoazobenzene. Both require polar electron-donating groups for binding to enzyme and are differentiated by their structure, their redox potentials, their rates of chemical and enzymic reduction, and the influence on their metabolism of inducing agents, CO and O2. Azo compounds whose reductions are insensitive to CO and O2 (I-substrates) contain electron-donating substituents on either ring. Azo compounds whose reductions are O2- and CO-sensitive (S-substrates) also contain electron-withdrawing groups on the opposite (prime) ring. For all dyes, NMR studies revealed minor differences in the chemical shifts of the protons attached to the phenyl ring substituted with electron-donating substituents (ring A). This is consistent with the narrow range of pKa's (basicity) and KM values for all substrates. However, there are significant differences in the chemical shifts of the aromatic protons of the prime ring (ring B). The difference in chemical shifts is most pronounced for aromatic protons adjacent to the prime ring substituents, showing a clear distinction between I and S substrates. Furthermore, the Hammett sigma substituent constants on the prime ring clearly distinguish between the two classes of dyes. I- and S-substrates have negative and positive sigma Hammett values, respectively. This implies that the mechanism of microsomal azoreduction is critically dependent on the charge and redox potentials of the dyes and is exclusively determined by the nature of the substituents on the prime ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Hepatic microsomal azoreductase activity. Reactivity of azo dye substrates is determined by their electron densities and redox potentials.

We have previously established that microsomal reduction of azo dyes requires polar electron-donating ring substituents [e.g. -OH, -NH2, -NHCH3, or -N(CH3)2]. Reduction of dyes substituted exclusively with such moieties on either phenyl ring is insensitive to both O2 and CO (I-substrates). In contrast, azoreduction of compounds containing additional electron-withdrawing substituents (e.g. -SO3H, -COOH, -COOCH3, and -AsO3H2) on the opposite (prime) phenyl ring is sensitive to both O2 and CO (S-substrates). We have recently shown that Hammett aromatic substituent constants and redox potentials of the dyes, determined by cyclic voltammetry (CV), distinguish between I- and S-substrates. Dyes that exhibit positive Hammett sigma substituent constants on 1 of the 2 rings are S-substrates, and undergo immediate quenching of their one electron-reduced intermediates upon exposure to air, as observed by CV. In contrast, dyes that have negative Hammett sigma values on one or both rings are I-substrates, and their one electron-reduced intermediates are relatively stable in air. In this study, we have investigated the susceptibility to microsomal azoreduction of monosubstituted dyes, with ring substituents possessing a wide range of Hammett sigma values. We have observed a direct correlation between the susceptibility of these compounds to microsomal azoreduction and the Hammett sigma constant of the aromatic ring substituent, and the presence of a positive potential in the dyes observed by CV. A Hammett sigma value of -0.37 or lower is required for substrate activity. Dyes with two substituents in the prime ring confirmed the previous correlation of negative and positive Hammett sigma values with I- and S-substrate activities, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗