Structural studies of α-crystallin

1. alpha-Crystallin has been isolated from the cortex of ox lens by isoelectric precipitation followed by chromatography on DEAE-cellulose. The amino acid composition is in agreement with that reported for alpha-crystallin prepared by a different method. There is one thiol group/20000g. of protein (20000 is the order of magnitude of the sub-unit molecular weight), and disulphide bonds are absent. 2. The thiol group has been alkylated with radioactive iodoacetate in the presence of urea. 3. Partial acid hydrolysis of the alkylated protein gives, according to the conditions, mainly three radioactive peptides or nearly exclusively one radioactive dipeptide. The dipeptide is N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys. The two other peptides are probably the tripeptides related to Ser-CMCys. 4. The simplest interpretation of these results is that the sequence around the cysteine residue is a common structural feature of the sub-units of alpha-crystallin.     

How α-crystallin prevents the aggregation of insulin

Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with alpha-crystallin in solution were studied. Prevention of the aggregation of insulin by alpha-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with alpha-crystallin. The conformational changes in alpha-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from alpha-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

N-Terminal sequences of α-crystallin

The amino acid sequences at the N-terminal ends of the chains of the lens protein, alpha-crystallin, were studied. Both the main kinds of chain in bovine alpha-crystallin (A chains and B chains) have an N-terminal methionine residue, and the amino group is acetylated. Selective purification of the peptides in a tryptic digest of bovine alpha-crystallin gave a preparation consisting largely of the N-terminal peptide from the A chains, and the sequence of this peptide was elucidated. Subsequently, the N-terminal peptides were prepared from separated A and B chains. The proposed sequences are: A chain, acetyl-Met-Asp-Ile-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys; B chain, acetyl-Met-Asp-Ile-Ala-Ile-His-(Pro,Trp)-Ile-Arg. The similarity between the sequences supports the hypothesis that the A and B chains are derived evolutionarily from a common precursor.     

Further studies of the β-lactamases ofBacteroides species

The -lactamases of individual strains ofBacteroides fragilis, B. thetaiotaomicron, andB. melaninogenicus were examined to characterize their enzymatic activity and the relation between the periplasmic and cytoplasmic forms of the enzymes. K_m and V_max values indicate that all strains examined were very similar in terms of enzymatic activity with the antibiotics tested. Electrophoretic analysis and treatment with phospholipase D suggest the presence of a cytoplasmic form of the enzyme that is modified upon entry into the periplasmic space.     

Kinetic studies on the loss of water from α-D-glucose monohydrate

Although the dehydration of α-D-glucose monohydrate is an important aspect of several industrial processes, there is uncertainty with regard to the applicable rate law and other factors that affect dehydration. Therefore, the dehydration of three glucose monohydrate samples has been studied using isothermal gravimetric analysis. Dehydration follows a one-dimensional contraction (R1) rate law for the majority of kinetic runs, and an activation energy of 65.0±3.9 kJ mol(-1) results when the rate constants are fitted to the Arrhenius equation. Fitting the rate constants to the Eyring equation results in values of 62.1±3.7 kJ mol(-1) and -77.8±4.7 J mol(-1)K(-1) for ΔH(?) and ΔS(?), respectively. The impedance effect on the loss of water vapor has also been investigated to determine the values for activation energy, enthalpy, and entropy for diffusion of water. The results obtained for the activation parameters are interpreted in terms of the absolute entropies of anhydrous glucose and the monohydrate.     

The molecular weight of the basic polypetide chain αB2 of α-crystallin

The molecular weights calculated from the amino acid sequences of the A and B chains of the lens protein -crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for A and 22500 for B. The discrepancy between the value of 22500 and the real molecular weight of 20070 for B vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.     

Binding of γ-crystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone α-crystallin

α-Crystallin is a small heat shock protein and molecular chaperone. Binding of Cu2+ and Zn2+ ions to α-crystallin leads to enhanced chaperone function. Sequestration of Cu2+ by α-crystallin prevents metal-ion mediated oxidation. Here we show that binding of human γD-crystallin (HGD, a natural substrate) to human αA-crystallin (HAA) is inversely related to the binding of Cu2+/Zn2+ ions: The higher the amount of bound HGD, the lower the amount of bound metal ions. Thus, in the aging lens, depletion of free HAA will not only lower chaperone capacity but also lower Cu2+ sequestration, thereby promoting oxidation and cataract.     

Further observations on the activation of lysosomal acid α-glucosidase by cortisone derivatives

1. Cortisone acetate activates the acid alpha-glucosidase in rat liver slices and in isolated liver lysosomes. 2. The reaction is steroid specific and moreover does not occur with lysosomal acid phosphatase or beta-galactosidase. 3. After pretreatment of the lysosomes with cortisone, substrate (maltose) binding to the soluble lysosomal acid alpha-glucosidase is not affected, but the steroid does increase the V(max.) value. Membrane-bound enzyme is not activated by cortisone. 4. 4-[(14)C]Cortisone is preferentially bound to the lysosomal membrane and the possible involvement of this structure in the activation phenomenon is discussed.     

Calcium-induced decrease of the thermal stability and chaperone activity of α-crystallin

α-Crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. α-Crystallin aggregation is involved in lens opacification, and high [Ca²⁺] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca²⁺ on the thermal stability of α-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca²⁺-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca²⁺] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca²⁺ concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca²⁺ decreases both secondary and tertiary–quaternary structure stability of α-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca²⁺ alters the structural stability of α-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, α-crystallin aggregation facilitated by Ca²⁺ would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Specific dissociation of αB subunits from α-crystallin

Exposure of bovine α-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the αB chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only αA chains. The decrease in the molar mass corresponds to the mass of the αB chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo α-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for α-crystallin and 4.0 for a homopolymer constructed from purified αB₂ polypeptides. An αA₂ homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The αB chains were found to be completely denatured, whereas the structure of the αA chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the αB chain was responsible for the specific dissociation of this polypeptide.     

Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Does the crowded cell-like environment reduce the chaperone-like activity of α-crystallin?

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.     

Further quasi-classical trajectory studies on the C++ H2O reaction

Further trajectory studies on the C⁺ + H₂O reaction have been performed using a potential energy surface described through a finite element method in its p version. In former trajectory studies [Y. Ishikawa, T. Ikegami and R.C. Binning Jr., Direct ab initio molecular dynamics study of C⁺+H₂O: angular distribution of products and distribution of product kinetic energies, Chem. Phys. Lett. 370 (2003), pp. 490–495; J.R. Flores, Quasichemical trajectories on a finite element density functional potential energy surface: the C⁺+H₂O reaction revisited, J. Chem. Phys. 125 (2006), 164309], tunnelling was not taken into account. The present results together with the analysis of the electronic excited states [J.R. Flores and A.B. González, The role of the excited electronic states in the C⁺+H₂O reaction, J. Chem. Phys. 128 (2008), 144310] are useful to interpret the mechanism of the title reaction, which has been the subject of crossed beam experiments [D.M. Sonnenfroh, R.A. Curtiss and J.M. Farrar, Collision complex formation in the reaction of C⁺ with H₂O, J. Chem. Phys. 83 (1985), pp. 3958–3964] and can be considered a prototypical ion–molecule reaction.     

Further family studies on the genetic control of β2-glycoprotein I concentration in human serum

Summary 88 families with a total of 213 children were examined for ₂-glycoprotein I serum concentrations. In 74 families parents and children had normal concentrations. In 9 families one of the parents and approximately half of the children had intermediate concentrations. These individuals are presumably heterozygous for a deficiency gene Bg^D. In these families ₂-glycoprotein I concentration appears to be controlled by a pair of alleles which are transmitted as autosomal co-dominants. The results in 5 families did not conform to this genetic hypothesis, since children with an intermediate concentration of ₂-glycoprotein I were found whose both parents had a normal concentration of this protein. Non-genetic factors may be responsible for phenotypic variations in the different genetic types.Supported by U.S.P.H.S. Grant AM 11796-02 and by the Deutsche Forschungsgemeinschaft.