Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains. (1/38)

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.  (+info)

Phosphate transport in rat liver mitochondria. Membrane components labeled by N-ethylmaleimide during inhibition of transport. (2/38)

N-ethylmaleimide (NEM) inhibits the transport of phosphate in mitochondria but is without effect on permeation of other metabolities. In spite of its specificity for inhibition of phosphate transport, NEM reacts in an unspecific manner with inner membrane proteins in general. Treatment of mitochondria with [3H]NEM just sufficient to produce inhibition of phosphate transport results in labeling of at least 10 polypeptide components of the inner membrane. A marked increase in the specificity of reaction of NEM for components of the phosphate transport system is attained by first protecting the transport system with p-mercuribenzoate (p-MB) and then by irreversibly blocking reactive sulfhydryl groups unassociated with transport by the addition of unlabeled NEM. Subsequent addition of dithiothreitol removes p-MB and restores 65 to 75 percent of the original phosphate transport activity. Reinhibition of transport with [3H]NEM results in both a 6-fold decrease in the amount of [3H]NEM bound by purified inner membrane vesicles and a substantial reduction in the number of labeled polypeptide components. Five distinct labeled species are detected by this method, one of which is a 32,000 molecular weight protein containing 40 percent of the bound radioactivity, or approximately 160 pmol/mg of inner membrane protein. Correlation of binding of [3H]NEM by inner membrane proteins with inhibition of phosphate transport suggests that the maximum concentration of the NEM-sensitive component of the phosphate transport system is 60 pmol/mg of mitochondrial protein. This value, when combined with V-max of NEM-sensitive transport of 205 nmol times min-1 times mg-1 at O degrees (Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249, 2593) yields an approximate minimum turnover for this process of 3500 min-1 at 0 degrees. This turnover number is at least 20-fold greater than similarly calculated values for adenine nucleotide transport and succinate oxidation in rat liver mitochondria at this temperature. Taken together these results suggest that the NEM-sensitive phosphate transport system in rat liver mitochondria has an unusually high catalytic activity compared to other mitochondrial processes, and that at least one of the five NEM-binding proteins is likely to be an essential component of this transport system.  (+info)

Sliding of the epithelium in experimental corneal wounds. (3/38)

The corneal epithelial cell has a unique sliding capability. The epithelial cell spreads and migrates in an amebic fashion without mitotic activity when the continuity of the epithelium is broken. This movement is demonstrated both in vivo and in vitro. Prompt sliding for sealing the wound defect is apparently the first step of the wound healing of the superficial cornea. Cut edges of collagen fibers show no sign of activity towards healing the wound. The energy source of the sliding is provided mainly from stored glycogen in the epithelial cells. Sliding is inhibited by removal of glycogen from the cell or by adding glycolytic enzyme inhibitors.  (+info)

Subunit structure of anthranilate synthetase from Neurospora crassa. (4/38)

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.  (+info)

Free fatty acids associated with the high molecular weight aminoacyl-tRNA synthetase complex influence its structure and function. (5/38)

Aminoacyl-tRNA synthetases from higher eukaryotes often are isolated as high molecular weight complexes associated with other components such as lipids. Since hydrophobic interactions are involved in the organization of the complex, it has been suggested that interaction of synthetases with these lipids might be important for their structure and function. Delipidation is known to affect certain properties of synthetases within the complex including sensitivity to detergents plus salts, temperature inactivation, hydrophobicity, sensitivity to proteases, and, as shown here, sensitivity to p-mercuribenzoate and sites of papain cleavage. Of the lipids known to co-purify with the complex, cholesterol esters, phospholipids and free fatty acids, we show that the particular lipids responsible for many of these changes are the free fatty acids. Specific removal of fatty acids results in a complex with properties similar to one totally delipidated by detergent treatment, and readdition of the fatty acid fraction reverses the effects. The fatty acid fraction contains both saturated and unsaturated fatty acids, but unsaturated fatty acids are much more effective in reversing the properties of the delipidated complex that are saturated fatty acids. These results indicate that the free fatty acids co-purifying with the synthetase complex bind to the synthetases and affect their structure and function.  (+info)

Conformation and spin state in methemoglobin. (6/38)

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.  (+info)

Observations on the mechanical precipitation of oxy Hb S and other mutants. (7/38)

Oxyhemoglobin S exhibits greater mechanical instability than oxyhemoglobin A. The rate of precipitation of Hb S when agitated by vortexing depends upon the geometry of the tube, the volume of the hemoglobin solution, and the concentration of hemoglobin. The rate of precipitation is inversely related to concentration. Precipitation is inhibited by temperatures near 4 degrees C and alkylureas whose protective capacity is approximately proportional to the carbon chain length of the alkyl group. Blocking the beta93 -SH group with parahydroxymercuribenzoate has only a small enhancing effect on the precipitation rate. Other mutants such as Hb Gun Hill, Leiden, (both heat unstable), and C-HARLEM are also unstable. In the case of C-HARLEM, the precipitation rate is greater than that for Hb S. The heat-unstable mutants are not as well protected by cold temperatures or alkyl ureas. D2O has only a minor stabilizing effect on hemoglobin S, but NaCl and related salts markedly enhance precipitation at concentrations of 0.5 M. It is concluded that mechanical instability of oxyhemoglobins is a multifactorial process involving surface denaturation, pH, ionic strength, hydrophobic interactions, protein conformation, and primary protein structure. This phenomenon will require more extensive investigation.  (+info)

Properties of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductase from pig kidney. Amino acid composition, reactivity of cysteinyl residues, and stereochemistry of D-glyceraldehyde reduction. (8/38)

Some physical and chemical properties of the monomeric NADP+-dependent aldehyde reductase (previously called TPN-L-hexonate dehydrogenase or D-glucuronate reductase) from pig kidney have been examined. The amino acid composition has been determined. Four of the five thiol groups react with p-mercuribenzoate at pH 7, with no resulting loss of catalytic activity. High concentrations of p-mercuribenzoate cause complete enzyme inhibition, which can be partly reversed by addition of aldehyde reductase is low (9%, estimated from the ellipticity at 208 nm), and 70 to 80% of the tyrosine and tryptophan residues aare buried within the molecule. One molecule of NADPH binds to the enzyme (Kp equal 25 muM), causing a blue shift and enhancement of the coenzyme fluorescence, and suggesting that the environment of the active site is hydrophobic. In the reduction of D-glyceraldehyde, catalyzed by aldehyde reductase, the pro-4R "A" hydrogen of NADPH attacks the re face of the carbonyl group. This stereospecificity is the same as in the reductions of D-glyceraldehyde and acetaldehyde effected by rabbit muscle dehydrogenase and liver alcohol dehydrogenase, respectively.  (+info)