Influence of tilt and decentration of scleral-sutured intraocular lens on ocular higher-order wavefront aberration.

AIM: To investigate the influence of tilt and decentration of scleral-sutured intraocular lenses (IOLs) on ocular higher-order wavefront aberrations. METHODS: In 45 eyes of 36 patients who had undergone scleral suture fixation of posterior chamber IOL, tilt and decentration of IOLs were determined by Scheimpflug videophotography, and higher-order aberration for a 4-mm pupil was measured using the Hartmann-Shack aberrometer. In another 100 eyes of 100 patients after standard cataract surgery with posterior chamber IOL implantation, ocular higher-order aberration was measured. RESULTS: In eyes with scleral-sutured IOL, the mean (SD) tilt angle and decentration were 4.43 degrees (3.02 degrees ) and 0.279 (0.162) mm, respectively. Ocular coma-like aberration in the sutured IOL group was 0.324 (0.170) microm, which was significantly greater than that of the standard cataract surgery group (0.169 (0.061) microm, p<0.001, Student's t test). No significant difference was found in ocular spherical-like aberration between the sutured IOL group (0.142 (0.065) microm) and standard surgery group (0.126 (0.033) microm; p = 0.254). In the sutured IOL group, IOL tilt significantly correlated with ocular coma-like aberration (Pearson's correlation coefficient r = 0.628, p<0.001), but no significant correlation was found between IOL tilt and ocular spherical-like aberration (r = 0.222, p = 0.175). The IOL tilt did not correlate with corneal coma-like (r = 0.289, p = 0.171) and spherical-like (r = 0.150, p = 0.356) aberrations. The IOL decentration did not correlate with any higher-order aberrations. CONCLUSION: In eyes with scleral-sutured posterior chamber IOL, tilting of the lens induces considerable amount of ocular coma-like aberrations.

On the specific association of porcine erythrocyte catalase caused by formation of disulfide cross-links.

Porcine erythrocyte catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified from porcine blood by DEAE-cellulose column chromatography, ammonium sulfate fractionation and CM-cellulose column chromatography. The purified enzyme was found to associate into larger molecules than the native one when it was stored at 4degrees C for more than one week. The associated molecules can be detected by gel filtration on a Bio-gel A-1.5 m column and disc gel electrophoresis as well as ultracentrifugal analysis. Molecular weights of the associated catalase molecules were about 500 000, 750 000, 1 000 000 and so forth estimated by gel filtration and disc gel electrophoresis, corresponding to dimer, trimer and tetramer respectively, of a native molecule (monomer) with a molecular weight of about 250 000. The association of catalase molecules is found to be time-dependent and to proceed seemingly from monomer through dimer as an intermediate. From the effects of several thiol reagents or reducing reagents on the association process and spectrophotometric titration of SH groups, it is inferred that this specific association of procine erythrocyte catalase is caused by formation of intermolecular disulfide cross-links due to air oxidation of SH groups in the protein moiety.

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.

Comparison of the gamma-crystallins isolated from eye lenses of shark and carp. Unique secondary and tertiary structure of shark gamma-crystallin.

gamma-Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate gamma-crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark gamma-crystallin lie in the fact that this crystallin possessed a significant alpha-helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous gamma-crystallins from other vertebrate species including bony fishes which all show a predominant beta-sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of gamma-crystallins. N-Terminal sequence analysis corroborates the structural differences between shark and carp gamma-crystallins. gamma-Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian gamma-crystallin.

The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation.

Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.

Subcellular distribution and characterization of porcine kidney catalase.

Subcellular distribution of catalase for porcine kidney was analyzed by differential centrifugation of kidney homogenate. The specific activity of catalase was the highest in light mitochondrial fraction, followed by mitochondrial, cytosolic, nuclear, and microsomal fractions. However, about a half of the total activity was found in supernatant (cytosol) fraction and the other half was mainly associated with both mitochondrial and light mitochondrial fractions. Osmotic shock using hypotonic solution, 50 mm sodium phosphate buffer (pH 7.0) was found to be most effective for solubilization of particulate-bound catalase. Both particulate and cytosol catalases from porcine kidney were purified by ammonium sulfate fractionation followed by DEAE-cellulose, CM-cellulose, and Sephadex G-100 column chromatographies. The purified enzymes showed two distinct bands, one major and the other minor, on disc gel electrophoresis. Both particulate and cytosol enzymes showed identical molecular weights estimated from disc gel electrophoresis; that of the major component was 219,000 corresponding to native molecule and that of the minor one 421,000. A similar value, 210,000, was also obtained for the major component by gel filtration on a Bio Gel A-1.5 m column. It was inferred that the minor component was formed by dimerization of native molecule caused by formation of disulfide cross-links due to oxidation of SH groups in protein moiety. The particulate and cytosol catalases showed essentially identical molecular characteristics, although a slight difference was detected in stability in guanidine-HCl solution. The effect of NaCl on enzyme activity and optical properties of catalase were also measured.

Effects of halide ions on porcine kidney catalase.

The inactivating effects of halide ions on porcine kidney catalase were investigated. It was found that the inactivation of catalase was dependent on incubation time with halides and on their concentrations: the addition of 2 M NaCl reduced the activity to 18% of the original level, whereas the same extent of inactivation was obtained in the presence of 0.1 M NaF. Removal of excess halide ions by dialysis resulted in good recovery of enzyme activity for all halides examined except for KI. Additions of halide ions to catalase solution caused subtle but evident alterations in the absorption and CD spectra. We could detect clear difference spectra between the salt-treated and native catalase solutions. These difference spectra showed halide concentration dependence with an evident isosbestic point. From these changes and also changes in CD spectra, we deduced that fluoride, chloride, and bromide ions can bind with heme iron of the catalase molecule as ligands to form stable catalase-halide complexes, but iodide ions showed a different reactivity with catalase from other halides and may cause gross alteration in the structure or conformation of catalase. Dissociation constants (Kd) were estimated to be 2.5, 0.23, and 26 M for chloride, fluoride, and bromide complexes with catalase, respectively, and there is no heme-heme interaction during formation of the catalase-halide complexes as estimated from the Hill plot.

Isolation and characterization of two thiol proteinase inhibitors of low molecular weight from newborn rat epidermis.

Two low-molecular-weight thiol proteinase inhibitors, TPI(1) and TPI(2), were purified from newborn rat epidermis by homogenization and extraction at 37 degrees C, followed by lyophilization, Sephadex G-75 gel filtration and DEAE-cellulose column chromatography. Both purified inhibitors were shown to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of SDS. TPI(1) was more anionic than TPI(2), and the molecular weights of TPI(1) and TPI(2) were determined to be ca. 12,000 and 13,000, respectively. They were highly specific for thiol proteinase; TPI(1) was especially more effective than TPI(2) on the proteolytic activity of casein and hydrolytic activity of papain on authentic substrates, and a similar but lesser effect was detected for ficin. They inhibited the enzyme in a noncompetitive manner with inhibition constants of 6.01 X 10(-8) M and 4.57 X 10(-7) M for TPI(1) and TPI(2), respectively, calculated from the BAPA hydrolytic activity of papain. Both inhibitors were quite stable proteins as to heat and extreme pHs. The amino acid composition of TPI(1) and TPI(2) were slightly different from each other, and both inhibitors contained no cysteinyl or cytinyl residue and trptophanyl residue. From these results, TPI(1) and TPI(2) seem to be iso-inhibitors. The absorption and CD spectra of TPI(1) and TPI(2) in the near ultraviolet region indicated that the conformation in the vicinity of aromatic amino acid residues in TPI(1) was slightly different from that of TPI(2). The CD spectra of both inhibitors in the far ultraviolet region showed the presence of the beta turn structure in the protein moiety, suggesting some important roles of the beta turn of both inhibitors in the interaction with the thiol enzymes.

The interactions of thiol compounds with porcine erythrocyte catalase.

The effects of thiol compounds on the conformation of porcine erythrocyte catalase were examined. The thiol compounds showed two types of reactivity with the catalase in terms of changes in absorption spectra. One is characterized by the appearance of a new absorption maximum at 595 nm; this was seen with 2-mercaptoethanol (designated as inactive catalase type I). The other is characterized by new maxima at 535 and 570 nm, and this was seen with reduced glutathione, dithiothreitol, cysteine, and cysteamine (inactive catalase type II). The thiol compounds caused gradual inactivation of catalase, correlating with the enhancement of the absorption maximum at 595 nm or 570 nm. Removal of excess thiol reagents from the reaction mixtures caused partial recovery of activity, which was more marked with inactive catalase type II. Similar reversibility was observed in the absorption, CD and MCD spectra, whereas reversibility was not observed for inactive catalase type I. The MCD spectra suggested conversion of heme groups from a high to a low spin state on incubation with thiols, e.g., reduced glutathione, leading to inactive catalase type II. alpha-Helical conformation of the polypeptide backbone and titratable free SH groups in the catalase molecule were unaffected by all these thiol treatments. It is suggested that "active oxygen" which may be produced on incubation of catalase with thiol compounds, was responsible for the formation of inactive catalase type II.

On the denaturation of porcine erythrocyte catalase with alkali, urea, and guanidine hydrochloride in relation to its subunit structure.

The dissociation of porcine erythrocyte catalase [EC 1.11.1.6] into subunits on denaturation with alkali, GuHCl and urea was investigated by following the changes in hydrodynamic properties, absorption and CD spectra in the Soret region and inactivation of the enzyme. It was found that dissociation proceeded in an "all or none" manner from the native tetramer (molecular weight, ca. 250,000) into identical 1/4-sized monomers (molecular weight, ca. 54,000 with alkali, 65,000 with urea and 71,000 with GuHCl) as estimated by ultracentrifugal analyses. On this dissociation, the sedimentation coefficient decreased from about 11S to 5.1 - 3.7S, and absorption spectra in the Soret region decreased to about 40% of the native level and showed a broad band around 365-375 nm and a shoulder around 415-420 nm; these changes were accompanied by complete loss of enzyme activity. The change in enzyme activity correlated well with that of absorption and CD spectra in the Soret region, depending on denaturation time, alkaline pH used and concentration of both denaturants. The reassociated catalase obtained by removing urea by dialysis was characterized by recovery of distinct CD bands in the Soret and near ultraviolet regions, although the partial refolding of alpha-helical conformation occurred without recovery of enzyme activity. These results indicate that the conformational changes and dissociation process of catalase into subunits can be monitored spectrophotometrically in relation to enzyme activity, and that subtle conformations near the heme groups and polypeptide backbone play an important role in maintaining full enzyme activity of the catalase molecule.

DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography.

A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.

Primary structure of the inorganic pyrophosphatase from thermophilic bacterium PS-3.

The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes.

Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses.

Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli.

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.

Effects of divalent cations on thermophilic inorganic pyrophosphatase.

Divalent cations were shown to affect the structure and thermostability of thermophilic inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] purified from Bacillus stearothermophilus and thermophilic bacterium PS-3. The properties of the enzymes from the two sources were found to be very similar. The enzymes were very unstable to heart in the absence of divalent cations, being inactivated gradually even at 40 degrees C. However, they became stable to heat denaturation in the presence of Mg2+, between pH 7.8 and 9.0. Similar induced thermostability was detected when Mn2+, Co2+, Ca2+, Cd2+, and ZN2+ were added, though the latter three cations were not essential for enzyme activity. On adding divalent cations, the optical properties such as absorption spectra, fluorescence spectra, and circular dichroism (CD) were changed. Gel filtration and disc electrophoresis revealed that the molecular weight of both enzymes was 5.4 x 10(4) in Tris-SO4 buffer and 11 x 10(4) in Tris-HCL buffer, suggesting monomer-dimer transformation. In the presence of divalent cations in Tris-SO4 fuffer, the enzymes dimerized; this was confirmed by sedimentation velocity measurements. The enzymes in Tris-HCL buffer did not show thermostability unless divalent cations were added. The results in the present study indicate that binding of divalent cations to each enzyme caused some conformational change in the vicinity of aromatic amino acid residues leading to dimerization of the enzyme molecule so that it became thermostable. It was also suggested that histidyl residues play an important role in the thermostability induced by divalent cations on the basis of the pH dependencies of thermostability and CD spectra.

On the acid denaturation of porcine erythrocyte catalase in relation to its subunit structure.

Porcine erythrocyte catalase [EC 1.11.1.6] (molecular weight, ca. 250,000) was found to dissociate partially between pH 3.5 and 3.0 and completely below pH 3.0 into two presumably identical 1/2-sized subunits (molecular weight, 112,000) as estimated by ultracentrifugal analyses. This dissociation was accompanied by a marked change in hydrodynamic properties; the sedimentation coefficient decreased from about 11S to 4S. This acid denaturation also resulted in complete loss of enzyme activity and disappearance of absorption bands characteristic of heme protein, in particular, a shift of the Soret band from 405 nm to a small and broad band at 375 nm. The change in enzyme activity correlated well with that of the Soret band, depending on the denaturation time and pH used. Reversible recovery of enzyme activity was not detected below pH 3.1 after 2 h denaturation. The pH dependence of alpha-helical content estimated from the CD intensity at 222 nm also correlated well with that of enzyme activity. The rate constants of initial reaction of acid denaturation at several pHs were determined by following the changes in the Soret band with time, since the changes showed an isosbestic point at 384 nm. The results revealed that the first-order rate constant at pH 3.0 was 45 times larger than that at pH 3.4, indicating that the rate of acid denaturation increased rapidly within a narrow acidic pH range. The temperature dependence of the denaturation rate was also measured and the activation energy for the acid denaturation was found to be 76.3 kcal/mol from an Arrhenius plot.

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis.

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.

Hydrophobic interactions of Val75 are critical for oligomeric thermostability of inorganic pyrophosphatase from Bacillus stearothermophilus.

To determine the role of Val75 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) [EC 3.6.1.1] from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Val75 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp. As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability. The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V75I dissociated more slowly than the wild type. There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state. Thus, we conclude that Val75 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.

Comparative studies on the primary structure of human cystatin as from epidermis, liver, spleen, and leukocytes.

We have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes. These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition. The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A. The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides. Furthermore, the amino acid compositions of corresponding separated peptide quartets were identical. We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage. It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2-terminal methionine residue.