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.     

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.     

Immunoadsorption procedure as a potential method for the specific β2-microglobulin removal from plasma of patients with chronic renal failure

β₂-Microglobulin (β₂-M), which accumulates in the plasma of patients undergoing long-term dialysis, has been identified as the principal precursor protein of amyloid fibrils in dialysis-related amyloidosis. As no specific treatment for this affection has been yet established, an extracorporeal immunoadsorption procedure appears to be an attractive therapeutic approach to remove β₂-M. Several murine monoclonal antibodies to human β₂-M were developed and compared as affinity ligands. One of them was selected on the basis of its specificity and adsorption capacity. In order to achieve maximum efficiency in protein removal, different parameters of the procedure were studied and optimized: effect of antibody coupling density, determination of maximum adsorption capacity of the immunoadsorbents and influence of antigen concentration and of flow-rate on antigen capture efficiency. The conditions of regeneration of immunoaffinity sorbents were also investigated to allow their multiple use without loss of adsorption capacity. The results show the validity of the proposed technique in removing β-M from plasma of patients with chronic renal failure.     

An alternative splice variant of human αA-crystallin modulates the oligomer ensemble and the chaperone activity of α-crystallins

In humans, ten genes encode small heat shock proteins with lens αA-crystallin and αB-crystallin representing two of the most prominent members. The canonical isoforms of αA-crystallin and αB-crystallin collaborate in the eye lens to prevent irreversible protein aggregation and preserve visual acuity. α-Crystallins form large polydisperse homo-oligomers and hetero-oligomers and as part of the proteostasis system bind substrate proteins in non-native conformations, thereby stabilizing them. Here, we analyzed a previously uncharacterized, alternative splice variant (isoform 2) of human αA-crystallin with an exchanged N-terminal sequence. This variant shows the characteristic α-crystallin secondary structure, exists on its own predominantly in a monomer–dimer equilibrium, and displays only low chaperone activity. However, the variant is able to integrate into higher order oligomers of canonical αA-crystallin and αB-crystallin as well as their hetero-oligomer. The presence of the variant leads to the formation of new types of higher order hetero-oligomers with an overall decreased number of subunits and enhanced chaperone activity. Thus, alternative mRNA splicing of human αA-crystallin leads to an additional, formerly not characterized αA-crystallin species which is able to modulate the properties of the canonical ensemble of α-crystallin oligomers.     

Effects of r—HuEPO on the biophysical characteristics of erythrocyte membrane in patients with anemia of chronic renal failure

Using electron spin resonance (ESR) spin labeling technique,we have studied the conformation of sulfhydryl groups(-SH) binding sites in membrane proteins and mem brane fluidity of red blood cells(RBCs) from two groups of patients with anemia of chronic renal failure(ACRF).One of the groups is composed of patients who were untreated with recombinant human erythropoietin(r-HuEPO),and the other is composed of patients who were treated with r-HuEPO.The results indicated:1)the conformation of SH group binding site in RBC membrane proteins from former group was different from those of healty people.2)the fluidity in the region near the surface of RBC membrane from former group was lower than those of healthy people.3)However,the above biophysical properties of RBC membrane from later group were normal.We concluded that RBC membrane in patients with ACRF was abnormal,and the treatment of r-HuEPO may promote the production of normal RBCs,thus ameliorate the biophysical properties of RBCs from the patients with ACRF.     

Acute phase proteins are major clients for the chaperone action of α2-macroglobulin in human plasma

Extracellular protein misfolding is implicated in many age-related diseases including Alzheimer's disease, macular degeneration and arthritis. In this study, putative endogenous clients for the chaperone activity of α₂-macroglobulin (α₂M) were identified after human plasma was subjected to physiologically relevant sheer stress at 37 °C for 10 days. Western blot analysis showed that four major acute phase proteins: ceruloplasmin, fibrinogen, α₁-acid glycoprotein and complement component 3, preferentially co-purified with α₂M after plasma was stressed. Furthermore, the formation of complexes between α₂M and these putative chaperone clients, detected by sandwich ELISA, was shown to be enhanced in response to stress. These results support the hypothesis that α₂M plays an important role in extracellular proteostasis by sequestering misfolded proteins and targeting them for disposal, particularly during acute phase reactions.     

Ontogeny and localization of the α, β, and γ crystallins in newt eye lens development

The α-, β-, and γ-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the α-, β-, and γ-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. β-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. γ-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and α-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for β-crystallins until late in lens morphogenesis, and α- and γ-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.     

Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.     

In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna

Galactomannans are composed of a 1to4 mannan backbone with varying degrees of 1to6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased -galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of -galactosidase for the control of the final galactose content, a -galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing -galactosidases from germinating guar and coffee bean. The senna -galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R₁ plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna -galactosidase gene. This suggests that -galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Glutathione and γ-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats

Summary Components of the -glutamyl cycle, including thiols, glutathione (GSH) and -glutamyl transpeptidase (-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. -GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. Inrespiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, -GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the -glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.     

Purification and properties of α-d-mannosidase from the limpet, Patella vulgata

1. alpha-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. beta-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from beta-galactosidase. 2. Limpet alpha-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn(2+) for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn(2+). 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn(2+). 4. EDTA accelerated inactivation and the addition of Zn(2+) at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl(-) had an unspecific effect on hydrolysis by limpet alpha-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that alpha-mannosidase is a metalloenzyme or enzyme-metal ion complex, dissociable at the pH of activity, and that it requires Zn(2+) specifically.     

Cloning,expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m of 3.5?×?10⁷?M^?1 ×s^?1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.     

Rat α-foetoprotein. Purification, physicochemical characterization, oestrogen-binding properties and chemical modification of the thiol group

1. Rat alpha-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total alpha-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (K(a)=4.3x10(7)+/-1.2x10(7)m(-1)). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of alpha-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4'-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of alpha-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.     

Increased levels of plasma 8-EPI-PGF2α in patients with end stage renal failure

Summary

F₂-isoprostanes are a series of prostaglandin-F₂ like compounds specifically derived from peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi PGF_2α is shown to be a potent vasoconstrictor. In this study, we have analysed plasma 8-epi PGF_2α as a marker of oxidative stress in patients with end stage renal failure (ESRF) undergoing haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Plasma F₂-isoprostanes were isolated by solid-phase extraction on a C₁₈ followed by an NH₂ cartridge. Quantitative analysis of the F₂-isoprostanes as pentafluorobenzyl (PFB) ester/trimethylsilyl (TMS) ether derivatives was carried out by gas chromatography-electron capture mass spectrometry. For 34 individuals with ESRF, the mean level of esterified 8-epi PGF_2α was 0.58 ± 0.22 M; range 0.21–1.16 nM. 8-epi PGF_2α concentration in the patient groups was markedly higher (P<0.0005 by separate variance t-test) than that of control subjects (n=15) 0.28 ± 0.17 nM; range 0.02–0.63 nM. There was no difference in levels of 8-epi PGF_2α in plasma from patients undergoing HD or CAPD, nor was there any association with age, plasma lipids or plasma creatinine. These data provide direct evidence of increased oxidative stress in individuals with ESRF. This marker should be useful in clinical studies examining the degree of oxidative stress in vivo and the therapeutic impact of antioxidants.     

Purification,characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major β-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes β-1,3-glucans as laminarin and yeast β-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched β-1,3-glucans or mixed β-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.     

Diversification of Escherichia coli genomes: are bacteriophages the major contributors?

Determination of the genome sequence of enterohemorrhagic Escherichia coli O157 Sakai and genomic comparison with the laboratory strain K-12 has revealed that the two strains share a highly conserved 4.1-Mb sequence and that each also contains a large amount of strain-specific sequence. The analysis also revealed the presence of a surprisingly large number of prophages in O157, most of which are lambda-like phages that resemble each other. Based on these results, we discuss how the E. coli strains have diverged from a common ancestral strain, and how bacteriophages contributed to this process. We also describe possible mechanisms by which O157 acquired many closely related phages, and raise the possibility that such bacteria might function as 'phage factories', releasing a variety of chimeric or mosaic phages into the environment.     

Potent α-glucosidase inhibitors from the roots of Panax japonicus C. A. Meyer var. major

Bioassay-guided fractionation of the CHCl₃ soluble portion of the roots of Panax japonicus C. A. Meyer var. major afforded an active fraction with inhibitory activity against baker’s yeast α-glucosidase with an IC₅₀ value 1.02 mg/mL. Furthermore, the active fraction isolated contained three previously unreported polyacetylenes, designated panaxjapynes A–C, together with 11 other compounds, including four polyacetylenes, five phenolic compounds, a sesquiterpenoid, and a sterol glucoside. The structures of the compounds were elucidated by spectroscopic and chemical methods. Compared with the control acarbose (IC₅₀ 677.97 μM), six compounds were shown to be more potent α-glucosidase inhibitors with IC₅₀ values in the range 22.21–217.68 μM.