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.     

Age-related degradation of betaA3/A1-crystallin in human lenses.

The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.     

Metal-dependent proteinase of the lens. Assay, purification and properties of the bovine enzyme.

1. Two new assay methods were developed for the lens proteinase. In both, the substrate was alpha2-crystallin (a major lens protein); in the first method, the products were detected by reaction with trinitrobenzenesulphonate in the presence of SO32-, whereas in the second method, 3H-labelled substrate was used, and the products were detected as radioactivity soluble in trichloroacetic acid. 2. The neutral proteinase from bovine lens was partially purified by extraction of the lens at pH5.0 and column chromatography on hydroxyapatite and Sepharose 6B gel. 3. The purified enzyme had no detectable activity against haemoglobin, azo-casein or gamma-crystallin under optimum conditions for alpha2-crystallin. 4. The enzyme showed greatest activity and stability at pH7.5. It was reversibly inhibited by EDTA and 1,10-phenanthroline, and activated by Ca2+ and Mg2+. 5. Molecular weights obtained for the enzyme by chromatography on Sepharose 6B were approx. 500,000 in buffer of I = 0.02, and 250,000 at I = 1.02. 6. The properties of the purified lens proteinase are such as to suggest that this enzyme could account for the entire endopeptidase activity of the lens.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.     

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.     

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.     

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

A re-evaluation of the molecular size of duck ε-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of ε-crystallin isolated from the duck lens and lactate dehydrogenase of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of ε-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that ε-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and ε-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck ε-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50°C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

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.     

Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.     

In vivo heteromer formation. Expression of soluble betaA4-crystallin requires coexpression of a heteromeric partner

The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.     

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.     

BetaB2-crystallin undergoes extensive truncation during aging in human lenses

Based on the present literature, it is unclear whether betaB2-crystallin undergoes age-related truncation in human lenses. To answer this question, the purpose of this study was to determine in vivo truncation of betaB2-crystallin in human lenses during aging by examining its fragments in the beta(H)-crystallin fraction. The WS-protein fraction was isolated from lenses of desired ages and separated by a size-exclusion Agarose A 1.5m column to recover alpha-, beta(H)-, beta(L)-, and gamma-crystallin fractions. The beta(H)-crystallin fractions, isolated from lenses of 24- and 70-year-old donors, were utilized for two-dimensional (2D) gel electrophoresis (isoelectric focusing in the first dimension followed by SDS-PAGE in the second dimension). The partial N-terminal sequences of the desired fragments (Molecular weights [M(r)]<18-19kDa) from a 2D-gel of WS-proteins from lenses of a 70-year-old donor were determined. More than 37 crystallin fragments with M(r) between 4 and 19kDa were observed on a 2D-gel. Nine fragments in beta(H)-crystallin fraction were from betaB2-crystallin but additional single fragments of alphaA-, gammas-, betaA4, and of either gammaB-, gammaC- or gammaD-crystallins were also observed. Seven cleavage sites in the betaB2-crystallin were identified, which included two sites at Q(7)-A(8) and A(8)-G(9) bonds in the N-terminal extension, two sites at E(46)-K(47) and G(49)-S(50) bonds in the motif 1, one site at S(94) -S(95) in the motif 2, and two sites at N(115)-F(116) and Q(135)-Y(136) in motif 3. No fragments with cleavage in the motif 4 and C-terminal extension of betaB2-crystallin were seen. Apparently, three betaB2-crystallin fragments with only N-terminal cleavage and five with both N- and C-terminal cleavages were observed. Additional fragments with cleavage sites at Q(54)-Y(55) in alphaA-crystallin, at E(112)-N(113) in betaA4-crystallin, at G(4)-T(5) in gammas-crystallin, at M(69)-G(70) in either gammaB-, gammaC- or gammaD-crystallins (three have identical sequences at the cleaved bond), and at G(1)-K(2) in gammaB or gammaC (both have identical sequences at the cleavage site) were observed.Conclusions. The results showed that betaB2-crystallin undergoes age-related truncation producing fragments with M(r) between 4 and 19kDa that existed in the beta(H)-crystallin oligomer. The beta(H)-crystallin fraction also contained single fragments of alpha-, betaA4-, gammas-, and other gamma-crystallins.     

A low-calcium-requiring calcium-activated neutral proteinase from human placenta

A low-calcium-requiring calcium-activated neutral proteinase (mu CANP) has been purified to homogeneity from human placenta. The purification procedure includes chromatography on DEAE-cellulose, Ultrogel AcA-22 and DEAE-Sephadex in succession. The purified mu CANP is a thiol proteinase and requires calcium for activity. Half-maximal activation occurs at 40 microM calcium. It is a heterodimer with subunits of 74 kDa and 32 kDa. (The placental mCANP has subunits of 70 kDa and 32 kDa.) Mn2+ or Sr2+, in combination with Ca2+, activates the enzyme synergistically. The presence of both mCANP and mu CANP in equal proportion in human placenta is reported for the first time. This will facilitate a comparative study of these two forms of human calcium-activated neutral proteinase, especially their physiological structural and functional interrelationship. Maximal activation of the autolysed mCANP occurs at a calcium concentration much higher than that for mu CANP; and this autolysed mCANP does not cross-react with antiserum against mu CANP, suggesting that the two forms of proteinase are independent species.     

Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human gamma-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that betaA1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of betaA1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.     

Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, alphaB-(1-18) (MDIAIHHPWIRRPFFPFH) and betaA3/A1-(59-74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, gammaS-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact alpha-, beta-, and gamma-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of alphaB-(1-18) and betaA3/A1-(59-74) peptides increased the scattering of light by beta- and gamma-crystallin and alcohol dehydrogenase. The ability of alpha-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with alphaB-(1-18) and betaA3/A1-(59-74) peptides, whereas gammaS peptide had no effect on chaperone-like activity of alpha-crystallin. The betaA3/A1-(59-74 peptide caused a 5.64-fold increase in alphaB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in alphaB-(1-18) and betaA3/A1-(59-74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.     

Mutation in the betaA3/A1-crystallin encoding gene Cryba1 causes a dominant cataract in the mouse

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.     

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication     

High-yield purification of HIV-1 proteinase expressed by a synthetic gene in Escherichia coli.

A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed. The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes. The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein. Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates. These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site. The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography. A high yield and a high specific activity were achieved.