Protein-protein interactions among human lens acidic and basic beta-crystallins

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically. Proteins were also expressed for some physicochemical studies. The results indicate that all acidic-basic pairs (betaA-betaB) except betaA4-betaBs pairs show strong hetero-molecular interactions. For acidic or basic pairs, only two pairs (betaA1-betaA1 and betaA3-betaA3) show strong self-association. betaA2 and betaA4 show very weak self-association, which arises from their low solubility. Confocal fluorescence microscopy shows enormous protein aggregates in betaA2- or betaA4-crystallin transfected cells. However, coexpression with betaB2-crystallin decreased both the number and size of aggregates. Circular dichroism indicates subtle differences in conformation among beta-crystallins that may have contributed to the differences in interactions.     

Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin

Of the crystallin proteins of the lens, the principal subunit of the beta-crystallin, beta B2 (beta Bp), has been considered to be the only heat-stable protein because it does not precipitate upon heating. In our recent investigations, however, we have found that the alpha-crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100 degrees C. Using circular dichroism and fluorescence to monitor the conformational changes of alpha- and beta B2-crystallins upon heating, we found that alpha-crystallin maintains a high degree of structure, whereas the beta B2-crystallin shows a reversible sigmoidal order-disorder transition at about 58 degrees C.     

Rat lens beta-crystallins are internally duplicated and homologous to gamma-crystallins

The nucleotide sequence of two cloned rat lens beta-crystallin cDNAs pRL beta B3-2 and pRL beta B1-3 has been determined. pRL beta B3-2 contains the complete coding information for a beta-crystallin, designated beta B3, of 210 amino acid residues. pRL beta B1-3 is incomplete at its 5' end; the 5' codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRL beta B1-3 codes for a beta-crystallin polypeptide, designated beta B1, whose full length is 247 amino acid residues. Considerable sequence homology is noted between the amino- and carboxy-terminal halves of each protein. The two rat beta-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat gamma-crystallin (37%) and bovine and murine beta-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine gamma II-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the beta-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the gamma-crystallins.     

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.     

Isolation and characterization of cDNAs encoding beta A2- and beta A4-crystallins: heterologous interactions in the predicted beta A4-beta B2 heterodimer

Except for the two acidic chains, beta A2 and beta A4, the primary structures of all bovine beta-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both beta A2 and beta A4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. beta A2, comprising 197 aa, and beta A4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of beta A4 was made based on the coordinates of one subunit from the beta B2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine beta-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic beta-crystallins with a view to defining structural features necessary for subunit recognition in beta-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine beta-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic beta-crystallins.     

Association properties of betaB1- and betaA3-crystallins: ability to form heterotetramers

As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.     

Beta B1 crystallin is an amine-donor substrate for tissue transglutaminase

The effects of tissue transglutaminase on the water-soluble proteins in bovine lens homogenates are described. Addition of liver transglutaminase and Ca2+ to calf lens homogenates resulted not only in the appearance of 50- and 57-kDa dimers, but also in a decrease in the amount of beta B1 crystallin and the almost complete disappearance of beta B3 and beta A3. This is not the result of Ca2+-induced proteolysis, since histamine completely inhibits this phenomenon. It may be concluded that these polypeptides are involved in beta-crystallin crosslinking by transglutaminase. This notion was confirmed by using beta B1- and beta Bp-specific antisera. Both sera reacted with the 57-kDa dimer; the beta Bp-specific antiserum also reacted with the 50-kDa dimer. No reaction in the region 50-57 kDa was detectable when EDTA was used instead of Ca2+. Using reconstituted mixtures of beta B1- and beta Bp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the beta B1/beta Bp dimer, glutamine residue -9 of beta Bp crosslinks to one of the lysine residues in the N-terminal extension of beta B1.     

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions.

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.     

Evidence for the extralenticular expression of members of the β-crystallin gene family in the chick and a comparison with δ-crystallin during differentiation and transdifferentiation

The beta-crystallins are major water soluble proteins of vertebrate lens fibre cells and have previously been regarded as lens-specific proteins: however beta B2-and beta A3/A1-crystallin RNAs are transcribed and beta-crystallin polypeptides are detectable in the developing chick retina. The beta-crystallin RNA is transcribed in a subpopulation of retina cells and the number of transcribing cells and the level of beta-crystallin polypeptides increase during the differentiation of the retina. Several tissues express beta-crystallin polypeptides, but individual tissues are characterised by qualitative and quantitative differences in the beta- and delta-crystallin polypeptides expressed. The expression of beta-crystallins appears to be non-random as defined by tissue distribution, cellular localisation and ontogeny, implying a function for extralenticular beta-crystallins and a complex mechanism for the regulation of their expression.     

Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts.

The most prevalent proteins in the lens of the eye are called crystallins, and it is thought that aberrant crystallins may cause opacification of lens tissue. The Philly mouse, a strain with an inherited cataract, has an abnormal beta B2-crystallin, the principal beta-crystallin in the mouse. The cDNA that codes for the beta B2-crystallin protein has been cloned and sequenced from both the normal and the cataractous Philly mouse. The normal mouse beta B2 cDNA is 756 nucleotides in length with 618 nucleotides of open reading frame. An in-frame deletion of 12 nucleotides has occurred in the Philly mouse cDNA, which results in the loss of 4 amino acids. The sequence of the mutant beta B2 was analyzed against the reported structure of the normal bovine beta B2-crystallin determined by x-ray crystallography. The region, in which the deletion of the amino acids occurs near the COOH terminus, is essential for the formation of the tertiary structure of the beta B2-crystallin. The loss of these residues could explain the alterations that are seen with the Philly beta B2 protein and may account for the instability of the Philly beta B2 protein. This abnormal beta B2-crystallin may be the cause of the cataract in this animal.     

Subunit compositions of trypsin inhibitors from tubers of taro, Colocasia antiquorum var. nymphaifolia?

Several trypsin inhibitors with different mobilities on polyacrylamide gel electrophoresis occur in the tubers of taro (Colocasia antiquorum), and they each have a dimeric molecular weight of 40,000. Of all the constituent subunits, molecular weight 20,000, of the taro trypsin inhibitor (TTI), three major subunit components were separated by chromatography on SP-Sephadex C-25 in 8 M urea, and they were named protomers alpha, beta, and gamma in the order of their elution from the SP-Sephadex column. After removal or dilution of the urea, the three protomers could be either reassociated individually or hybridized with each other to form dimeric inhibitors. All of the reassociated dimers were powerful inhibitors of trypsin. Among them, each dimer derived from protomers alpha and gamma was a weak inhibitor of chymotrypsin, whereas the dimer of protomer beta did not inhibit the enzyme. Therefore TTI is presumed to be a mixture of heterogeneous and homogenous dimers whose properties reflect those of their constituent protomers. It was also proved that the major three trypsin inhibitors (TTI-I, TTI-II, and TTI-III) previously isolated from taro tubers are composed of protomers alpha and gamma, i.e., TTI-II is a heterogeneous dimer of protomers alpha and gamma, and TTI-I and TTI-III are homogeneous dimers of protomers alpha and gamma, respectively. The molecular weight of a trypsin-TTI complex saturated with trypsin was found to be 79,000, suggesting the formation of a tetrameric complex.     

Energetics of domain-domain interactions and entropy driven association of beta-crystallins

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.     

Acceptor-donor relationships in the transglutaminase-mediated cross-linking of lens beta-crystallin subunits

Following the isolation of the N epsilon-(gamma-glutamyl)lysine-containing polymers from human cataracts, our efforts were directed to induce such cross-links experimentally in rabbit lens, and evidence was obtained for the selective reactivities of certain beta-crystallin subunits in this transglutaminase-catalyzed event. In the present work, we examined the enzymatic cross-linking of purified crystallins individually (alpha, beta H, beta L, and gamma) and in combinations, with particular emphasis on forming the approximately 55K dimer. This species was the primary product in the cross-linking of beta H-crystallins; beta L also reacted with transglutaminase. Neither alpha- nor gamma-crystallins formed appreciable amounts of cross-linked structures with transglutaminase. Dansylcadaverine, known to compete against the reactive lysines of proteins in forming N epsilon-(gamma-glutamyl)lysine cross-bridges, was shown to inhibit the generation of dimeric and higher ordered oligomers from beta H and beta L. The fluorescent amine specifically labeled only two subunits in beta H (approximately 29-30K and approximately 26K) and one in beta L (approximately 26K), identifying these substrates as possessing transglutaminase-reactive endo-gamma-glutaminyl residues. An antiserum to bovine beta Bp recognized the approximately 23K subunit of rabbit beta-crystallins and also the approximately 55K dimer, suggesting that the approximately 23K protein participates as a lysine donor in generating the cross-linked dimer with transglutaminase. Inasmuch as the same antiserum reacts with an approximately 50K material reported to appear in increasing amounts with age in human lens, the results lend added support to the physiological significance of transglutaminase in the aging of lens.     

The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5-9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.     

Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers

The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.     

Purification, characterization, and amino acid composition of rabbit pulmonary bleomycin hydrolase

Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 mumol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56 degrees C, 10 h at 37 degrees C, and 5 days at 4 degrees C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI's of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.     

Calcium-binding to lens betaB2- and betaA3-crystallins suggests that all beta-crystallins are calcium-binding proteins

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. Betagamma-crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-crystallin for calcium-binding studies. Using calcium-binding assays such as 45Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both betaB2- and betaA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N-1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.