Physicochemical characterization of α-crystallins from bovine lenses: Hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HMα-crystallin and α-crystallins of bovine lens. Results from this study indicated that HMα (high-molecular-weight α-crystallin) and α (low-molecular-weight α-crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HMα with an average sedimentation coefficient of about 50 S and a more homogeneous system of α-crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant β-sheet conformation for α-crystallin, yet a highly asymmetrical and aggregated form for HMα. The conformational stability of α-crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native α-crystallin. Conformational flexibility of α-crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on α-crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HMα from α-crystallin. The comparison of conformational properties between HMα and α-crystallin strongly indicated that HMα is a denatured form of α-crystallin.     

Physicochemical characterization of γ-crystallins from bovine lens—Hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the γ-crystallin of the bovine lens. Sedimentation study indicates that γ-crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10^?7 cm²/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated γ-crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of γ-crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these γ-crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, γ-crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for γ-crystallins showed a minimum at about 217 nm, characteristic of a β-sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for γ-crystallin II.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Isolation and characterization of dioscin-α-l-rhamnosidase from bovine liver

A novel dioscin-α-l-rhamnosidase was isolated and purified from fresh bovine liver. The activity of the enzyme was tested using diosgenyl-2,4-di-O-α-l-rhamnopyranosyl-β-d-glucopyranoside as a substrate. It was cleaved by the enzyme to two compounds, rhamnoses and diosgenyl-O-β-d-glucopyranoside. The optimal conditions for enzyme activity were that temperature was at 42 °C, pH was at 7, reaction time was at 4 h, and the substrate concentration was at 2%. Furthermore, metal ions such as Fe³⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Mg²⁺ showed different effects on the enzyme activity. Mg²⁺ acted as an activator whereas Cu²⁺, Fe³⁺, and Zn²⁺ acted as strong inhibitors in a wide range of concentrations from 0 to 200 mM. It was interesting that Ca²⁺ played a role as an inhibitor when its concentration was at 10 mM and acted as an activator at the other concentrations for the enzyme. Moreover, the molecular weight of enzyme was determined as 75 kDa.     

Purification and characterization of β-xylosidase from Aureobasidium pullulans

Summary -Xylosidase was obtained from Aureobasidium pullulans CBS 58475 with an activity of 0.35 units/ml culture filtrate. The production of the enzyme was strongly inducible. -Xylosidase was purified in two steps by anion exchange and gel-permeation chromatography to high purity. The enzyme is a glycoprotein with an apparent molecular mass of 224 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates into two subunits of equal molecular mass. After SDS-PAGE -xylosidase could be renatured and stained with methylumbelliferyl--xylopyranoside. The enzyme was able to split substrates of other glycosidases. The maximum activity was reached at pH 4.5 and 80° C. -Xylosidase showed high stability over a broad pH range from pH 2.0 to 9.5 and up to 70° C. Analysis of cleavage patterns revealed that the enzyme was a typical glycosidase. Larger oligosaccharides consisting of xylose were degraded by an exomechanism together with a transxylosylation reaction.     

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that -defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine -defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with -defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel -defensin genes share the typical exon–intron structure and are highly homologous to published bovine -defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several -defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of -defensins from literature, the abundance of -defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that -defensins may play a role in local host defense during udder infections.The nucleotide sequence data reported in this article have been submitted to EMBL and have been assigned the accession numbers AJ563279–AJ563283, AJ567353–AJ567365, AJ567990–AJ567993, and AJ620296.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Purification and properties of β-ketothiolase from Zoogloea ramigera

-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of -ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits.The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent K _m value for acetoacetyl coenzyme A in the thiolysis reaction was 10 M, and that for coenzyme A was 8.5 M. The apparent K _m value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM.The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

Kinetics and properties of β-ketothiolase from Clostridium pasteurianum

1. Beta-Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis beta-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using beta-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. Beta-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA. 3. The beta-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 nM MgCl2. The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inihibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the beta-ketothiolases of C. pasteurianum and A. eutrophus were rather similar.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

The βγ-crystallins: Native state stability and pathways to aggregation

The βγ-crystallins are among the most stable and long-lived proteins in the human body. With increasing age, however, they transform to high molecular weight light-scattering aggregates, resulting in cataracts. This occurs despite the presence in the lens of high concentrations of the a-crystallin chaperones. Aggregation of crystallins can be induced in vitro by a variety of stresses, including acidic pH, ultraviolet light, oxidative damage, heating or freezing, and specific amino acid substitutions. Accumulating evidence points to the existence of specific biochemical pathways of protein: protein interaction and polymerization. We review the methods used for studying crystallin stability and aggregation and discuss the sometimes counterintuitive relationships between factors that favor native state stability and those that favor non-native aggregation. We discuss the behavior of βγ-crystallins in mixtures and their chaperone ability; the consequences of missense mutations and covalent damage to the side-chains; and the evolutionary strategies that have shaped these proteins. Efforts are ongoing to reveal the nature of cataractous crystallin aggregates and understand the mechanisms of aggregation in the context of key models of protein polymerization: amyloid, native-state, and domain-swapped. Such mechanistic understanding is likely to be of value for the development of therapeutic interventions and draw attention to unanswered questions about the relationship between a protein’s native state stability and its transformation to an aggregated state.     

ERRATUM: Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

We previously reported that the ocular lenses of the pontellid copepod Anomalocera ornata possess vertebrate-like β- and γ-crystallins. We cannot repeat our earlier data suggesting that the copepod lens crystallins belong to the β- and γ-crystallin family of proteins. Our new data are consistent with the copepod crystallins being novel proteins.     

Temperature-dependent coaggregation of eye lens αB- and β-crystallins

Crystallin is essential not only for the maintenance of eye lens transparency, but also in the biology of other tissues. Eye lens α-crystallin exists as a heteropolymer composed of two homologous subunits, αA and αB. Despite the critical role of α-crystallin in many tissues, little is known regarding structural and functional significance of the two subunits. Herein, we describe a unique feature of αB-crystallin. At high temperatures (>70 °C) not only αB-crystallin aggregates but also enhances the aggregation of other lens proteins. Intriguingly, αB-crystallin-mediated coaggregation at and above 70 °C involves β- but not γ-crystallin. Further, αA-crystallin, but not a mutant (F71L) αA-crystallin, prevented aggregation of αB-crystallin and also reduced coaggregation of αB- and β-crystallin. These studies explain the rationale for the existence of α-crystallin heteropolymer with αA subunit as a major partner that is vital for lens transparency and provide insights into αB-crystallin-induced coaggregation which may have a bearing in some pathological conditions where αB-crystallin is overexpressed.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.