Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.     

Protective effects of amino acids against freeze-thaw damage in mammalian cells.

Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins, a measure of hydrophobicity, is generally inversely proportional to the freeze-thaw protection by these free amino acids. However, the pattern of protection ("fingerprint") of cells by various amino acids is different from that of the enzymes liver alcohol dehydrogenase and calcium ATPase of the sarcoplasmic reticulum. Furthermore, unlike the case with these enzymes, for cells glutamine is the best cryoprotective agent of the amino acids tested.     

Crystallization of liver alcohol dehydrogenase activated by the modification of amino groups

Most of the amino groups of horse liver alcohol dehydrogenase were isonicotinimidylated with an imidoester. The enzyme activity increased about 17-fold. The modified protein crystallized during dialysis against buffers of low ionic strength in the monoclinic space group C2 with unit cell dimensions: $\text{a = 177.8}\text{A}\text{?}\text{, b = 61.2}\text{A}\text{?}\text{, c = 56.5}\text{A}\text{?}\text{and}\text{β = 104.0 °}$. The crystals appear to be suitable for detailed X-ray structure analysis.     

Anion binding to liver alcohol dehydrogenase

1. Complex formation at the general anion-binding site of the liver alcohol dehydrogenase subunit has been characterized by transient-state kinetic methods, using NADH as a reporter ligand. Equilibrium dissociation constants for anion binding at the site are reported. They conform basically to the lyotropic series of affinity order, with exceptionally tight binding of sulphate. The particular specificity for sulphate might be a general characteristic of anion-binding enzymic arginyl sites. 2. Anionic species of phosphate and pyrophosphate buffer solutions do not interact significantly with the general anion-binding site over the pH range 8-10. At lower pH, phosphate binding becomes significant due to complex formation with the monovalent H2PO4 species. The latter interaction corresponds to a dissociation constant of about 60 mM, indicating that phosphate binding is comparatively weak also at low pH. 3. It is concluded that previously reported pH dependence data for coenzyme binding to liver alcohol dehydrogenase cannot be much affected by coenzyme-competitive effects of buffer anion binding. Kinetic parameter estimates now determined for NADH binding in weakly buffered solutions agree within experimental precision with those obtained previously from measurements made in buffer solutions of 0.1 M ionic strength.     

Activation of liver alcohol dehydrogenase by glycosylation

D-Fructose and D-glucose activate alcohol dehydrogenase from horse liver to oxidize ethanol. One mol of D-[U-14C]fructose or D-[U-14C]glucose is covalently incorporated per mol of the maximally activated enzyme. Amino acid and N-terminal analyses of the 14C-labelled glycopeptide isolated from a proteolytic digest of the [14C]glycosylated enzyme implicate lysine-315 as the site of the glycosylation. 13C-n.m.r.-spectroscopic studies indicate that D-[13C]glucose is covalently linked in N-glucosidic and Amadori-rearranged structures in the [13C]glucosylated alcohol dehydrogenase. Experimental results are consistent with the formation of the N-glycosylic linkage between glycose and lysine-315 of liver alcohol dehydrogenase in the initial step that results in an enhanced catalytic efficiency to oxidize ethanol.     

Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped-flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2-chloroethanol, 2,2-dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6-10. Binary-complex formation between enzyme and alcohols leads to inner-sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc-bound water and the zinc-bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately by a factor of 10 on complex formation between enzyme and NAD+ or NADH. The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that enzyme-bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding. The pKa values determined for alcohol deprotonation at the binary-complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc-bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc-bound alcohols relatively to that of zinc-bound water.     

Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis

The dynamics of enzyme catalysis range from the slow time scale (～ms) for substrate binding and conformational changes to the fast time (～ps) scale for reorganization of substrates in the chemical step. The contribution of global dynamics to catalysis by alcohol dehydrogenase was tested by substituting five different, conserved amino acid residues that are distal from the active site and located in the hinge region for the conformational change or in hydrophobic clusters. X‐ray crystallography shows that the structures for the G173A, V197I, I220 (V, L, or F), V222I, and F322L enzymes complexed with NAD⁺ and an analogue of benzyl alcohol are almost identical, except for small perturbations at the sites of substitution. The enzymes have very similar kinetic constants for the oxidation of benzyl alcohol and reduction of benzaldehyde as compared to the wild‐type enzyme, and the rates of conformational changes are not altered. Less conservative substitutions of these amino acid residues, such as G173(V, E, K, or R), V197(G, S, or T), I220(G, S, T, or N), and V222(G, S, or T) produced unstable or poorly expressed proteins, indicating that the residues are critical for global stability. The enzyme scaffold accommodates conservative substitutions of distal residues, and there is no evidence that fast, global dynamics significantly affect the rate constants for hydride transfers. In contrast, other studies show that proximal residues significantly participate in catalysis.     

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of omega-hydroxylated fatty acids and steroids by alcohol dehydrogenase

Recrystallized alcohol dehydrogenase from horse liver was found to oxidize 17-hydroxystearic acid into 17-oxostearic acid, the 17-L-enantiomer faster than the 17-D-enantiomer. Alone at high pH or in combination with aldehyde dehydrogenase, the alcohol dehydrogenase also catalyzed conversion of 18-hydroxystearic acid into 1, 18-octadecadioic acid and 5β-cholestane-3α,7α,12α,26-tetrol into 3α,7α,12α-trihydroxy-5β-cholestanoic acid. All the activities as well as the ethanol dehydrogenase activity disappeared after specific carboxymethylation of a single cystein residue at the active site of alcohol dehydrogenase. These results conclusively show that alcohol dehydrogenase itself has ω-hydroxyfatty acid dehydrogenase activity and ω-hydroxysteroid dehydrogenase activity.     

Repair of oxidative DNA damage by amino acids

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)₂^–, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of ~10⁵, 10⁵, 10⁶ and 10⁷ dm³ mol^–1 s^–1, respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.     

Mechanism of freeze-thaw damage in Pseudomonas F8

Pseudomonas F8 was tested for freeze-thaw sensitivity after being washed several times with water. Survival increased to 85% in the fourth wash. Addition of 1% sodium chloride decreased survival to the level obtained when cells were frozen in brain-heart infusion broth. No differences were observed between cells grown at 5 or 23 °C. The freeze-thaw sensitivity of Pseudomonas may be due to the cell's exposure to concentrated medium salts during cooling and warming.     

Human alcohol dehydrogenase: dependence of secondary alcohol oxidation on the amino acids at positions 93 and 94.

The human liver alpha alpha and beta 1 beta 1 isoenzymes are straight-chain alcohol dehydrogenases with different efficiencies toward secondary alcohols. Two of the 24 amino acid substitutions in alpha alpha (A for F93 and I for T94) were made by site-directed mutagenesis of beta 1 beta 1 and the substrate specificity of beta 93A94I was examined. The Vmax/KM values of beta 93A94I for secondary alcohols (especially R enantiomers) are similar to that of alpha alpha and as much as 4000-fold greater than beta 1 beta 1, but the dependences of Vmax/KM on primary alcohol chain length are similar to beta 1 beta 1, but not alpha alpha. Thus, the substitutions of A for F93 and I for T94 in beta 1 beta 1 account for the increased efficiency towards secondary alcohols and stereoselectivity for enantiomeric alcohols, but not for the effects of chain length on the Vmax/KM for primary alcohols seen with alpha alpha.     

Intralobular distribution of rat liver aldehyde dehydrogenase and alcohol dehydrogenase

1. The activity of liver microsomal high Km-ALDH and mitochondrial low Km-ALDH, which may be primarily responsible for the oxidation of acetaldehyde after ethanol administration was found to be predominantly distributed in the centrilobular area. 2. The activities of other ALDH isozymes in mitochondrial and soluble fractions were evenly distributed in periportal and perivenous regions. 3. The activity of ADH which is involved in production of acetaldehyde was predominantly located in the periportal area. 4. From these results it seems unlikely that a concentration of acetaldehyde after ethanol ingestion is higher in perivenous hepatocytes than in periportal ones. Additional data would be needed to understand fully the mechanism by which ethanol induces predominantly centrilobular liver injury.     

Mechanism of liposome destabilization by polycationic amino acids

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-l-histidine and poly-l-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 11 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion.Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 °C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-l-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-l-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-l-histidine is observed over a range of temperatures from 0 to 40°C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation. We conclude that the promotion of leakage and fusion in anionic liposomes by polycationic amino acids is not a result of large changes in the physical properties or arrangements of the lipids but rather to a surface binding of the polyamino acids.Abbreviations DSC differential scanning calorimetry - DEPE dielaidoylphosphatidylethanolamine - POPS 1-palmitoyl-2-oleoylphosphatidylethanolamine - DMPS dimyristoylphosphatidylserine - DMPE dimyristoylphosphatidylethanolamine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine - T_H bilayer to hexagonal phase transition temperature - pyr-PE 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolaline - E/M ratio of intensities of excimer to monomer emission     

Raman spectroscopy of liver alcohol dehydrogenase

We report the Raman spectrum of liver alcohol dehydrogenase in solution. The enzyme's secondary structure as determined from an examination of the Raman bands is slightly different than that found in crystals by X-ray diffraction.