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Biomedical subjects

L Takemoto

Publications and source records attributed to L Takemoto.

At least 55 records · Page 3Linked to original sources
PubMed1991

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition.

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

Animals↗
PubMed1991

Characterization of the major cyanogen bromide fragment of alpha-A crystallin.

Alpha crystallin from the bovine lens has been digested with cyanogen bromide, and the major fragment (CB-1) has been purified using reverse phase HPLC. Characterization of this fragment by Edman degradation and antisera to synthetic peptides indicates that it originates from alpha-A crystallin, but lacks the N-terminal methionine and the last 35 amino acids from the C-terminus of the molecule. The purified CB-1 fragment binds as well as native alpha crystallin to lens membrane, but is unable to self-assemble into the correct size of high molecular weight oligomeric complexes characteristic of the intact alpha-A chain. Together, these results demonstrate that the alpha-A chain is comprised of at least two functional domains, one of which is involved in binding of alpha-A crystallin to lens membrane, and another which is necessary for correct self-assembly of the molecule into high molecular weight oligomers.

Amino Acid Sequence↗
PubMed1991

Age-dependent deamidation of the major intrinsic polypeptide from lens membranes.

The Major Intrinsic Polypeptide (MP26) of lens membranes contains an -asn-gly- sequence, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation. To determine if the asparagine residue of this sequence undergoes age-dependent deamidation in vivo, antiserum to a synthetic peptide containing the sequence was used to monitor purification of a tryptic peptide containing this sequence from fetal versus mature bovine lenses. The peptide from fetal lenses contained the -asn-gly- sequence, while the peptide from mature lenses contained an -asp-gly- sequence, demonstrating that age-dependent deamidation of this asparagine residue was occurring in the lens.

Aging↗
PubMed1991

Age-dependent covalent changes in MP18 from bovine lens membrane.

Peptide antisera made against the putative MP18 sequence recognizes an 18-kilodalton protein on western blots of bovine lens membrane. These peptide antisera were used to investigate possible age-related changes in the MP18 molecule in membrane fractions from newborn and adult bovine lenses. Western blot analysis of membrane fractions with peptide antisera to sequences 45-50 (anti-45), 113-129 (anti-113), and C-terminus 169-173 (anti-Ct) showed similar patterns. All three antisera reacted more strongly with MP18 from membrane prepared from newborn lenses than with corresponding preparations from the adult lens. Binding ratios of anti-45 to anti-Ct showed a statistically significant difference between cortex and nucleus of newborn lenses (P less than 0.02) and between cortex of newborn and cortex of adult lenses (P less than 0.001). Differences in binding ratios of anti-113 to anti-Ct were the most striking, with anti-113 unable to detect MP18 in membrane preparations from adult lenses. Collectively, these results confirmed the putative sequence of MP18 and demonstrated covalent changes in this molecule during the process of aging in the normal bovine lens.

Aging↗
PubMed1990

The sequences of two peptides from cataract lenses suggest they arise by deamidation.

Polyclonal antisera made against synthetic peptides corresponding to expected tryptic fragments of gamma crystallin have been used to screen tryptic digests of total proteins from cataractous versus normal human lenses. One of these antisera recognizes two peptides that were found in greater amounts from digests of cataractous lenses. These two peptides shared a common sequence that contained an aspartate residue in place of an expected asparagine, suggesting that increased deamidation of this residue had occurred in the human cataractous lens in vivo.

Aged↗
PubMed1990

Specificity of alpha crystallin binding to the lens membrane.

The A1, A2, B1, and B2 species of bovine alpha crystallin have been purified and renatured to form high molecular weight aggregates comprised of only one species, and the aggregated forms of each of these species have been tested for their ability to bind to lens membrane in vitro. The aggregated forms of alpha-A1 and alpha-A2 bound to membrane in a saturable manner while those of alpha-B1 and alpha-B2 bound in much lower amounts, in a manner inconsistent with saturable binding. Together, these results demonstrate specific and saturable binding of aggregated alpha-A1 and alpha-A2 to the lens membrane, suggesting that these species are responsible for the previously observed interaction between alpha crystallin and the lens fiber cell membrane.

Animals↗
PubMed1990

Covalent change in alpha crystallin in opaque and transparent sections from the same human cataractous lens.

Opaque and transparent regions from the cortex of the same human cataractous lens were removed, and the water soluble proteins from these sections were resolved by isoelectric focusing in the presence of 8M urea, followed by Western blot analysis using antisera to the whole alpha crystallin molecule. Analysis of 18 different sections from 3 different lens demonstrated statistically significant changes in the charge distribution of alpha crystallins between opaque and transparent sections, with the greatest changes occurring in the anterior, posterior and equatorial sections from a 66-year-old cataractous lens. All opaque sections from this lens contained alpha crystallin with more positive charge than the transparent sections from the same lens. This change was present in both the water soluble and the insoluble fractions of the microdissected sections, and could also be demonstrated by probing of the Western blots with antisera made against synthetic peptides corresponding to the N-terminal and C-terminal regions of the molecule. Together, these results demonstrate that during human senile cataractogenesis there is often a covalent modification of the alpha crystallin molecule, which is probably not due to simple proteolysis, and which results in a shift of the alpha crystallin molecule to a more basic form.

Amino Acid Sequence↗
PubMed1990

Antisera to alpha crystallin as probes to study changes in lens proteins during human cataractogenesis.

Antisera have been made to synthetic peptides that correspond to eight different regions of the alpha A molecule. Together with a solid phase radioimmunoassay, these antisera have been used to quantitatively assess binding to enriched alpha crystallin preparations from six different cataractous and six different normal lenses. Seven of the eight antisera show no difference in binding to alpha crystallin from cataractous versus normal lenses, whereas the antiserum directed against the alpha A sequence 120-130 shows a statistically significant decrease in binding to the alpha crystallin from cataractous lenses. Together, these studies demonstrate the feasibility of using antipeptide sera as probes of polypeptide changes during cataractogenesis and suggest that the region of the alpha A crystallin molecule encompassing residues 120-300 may undergo covalent and/or noncovalent structural modification during the process of opacification in the human senile lens.

Aged↗
PubMed1989

Antisera to synthetic peptides as probes of structural changes during aging of alpha-crystallin from the bovine lens.

Polyclonal antisera have been made to synthetic peptides of 11-15 residues that correspond to nine different regions of the alpha A crystallins. These antisera have been used in a radioimmunoassay to quantitatively probe for structural and/or covalent changes of alpha-crystallins in the nucleus versus cortex of the adult bovine lens. Antisera specific for the C-terminal and N-terminal regions of the alpha-crystallins bind more to alpha-crystallins from cortex. Antisera to three out of the seven internal sequences (residues 75-89, 87-101 and 135-149) bind better to alpha-crystallins from the bovine lens nucleus, suggesting a greater accessibility of these sequences to antisera binding. Together, these studies demonstrate that antisera against synthetic peptide sequences of alpha A crystallins are very specific probes that can detect structural and/or covalent changes in specified regions of the alpha-crystallins during the process of aging in the bovine lens.

Aging↗
PubMed1989

Immunochemical characterization of the major low molecular weight polypeptide (10K) from human cataractous lenses.

Polyclonal antisera to whole crystallins and to synthetic peptides corresponding to various sequences of these crystallins have been used to probe Western blots that contain a low molecular weight component of approximately 10,000 daltons found in the water-soluble fractions from human cataractous lenses. This 10K component binds only to antiserum made against human gamma crystallin. Incubation of human cataractous lens homogenates with alpha chymotrypsin or trypsin will produce low molecular components of similar molecular weight, and identical specificity of binding to the gamma crystallin antiserum. Together, these results suggest that the gamma crystallins constitute a class of macromolecules that are susceptible to in vivo proteolysis during cataractogenesis of the aged human lens.

Aged↗
PubMed1989

Covalent change in the major intrinsic polypeptide (MIP26K) during cataract development in the streptozotocin-induced diabetic rat.

Antisera to synthetic peptides corresponding to residues 229-237, 252-259, and 256-263 have been used to quantitatively bind to the 19.5K, 24.0K, and 26.5K forms of the Major Intrinsic Polypeptide (MIP26K) of lens membrane from the streptozotocin-induced diabetic rat. The binding ratio of anti-229/anti-252 for the 19.5K component, and the binding ratio of anti-252/anti-256 for the 26.5K component, both increase only during the opacification process of the diabetic lens. Together, these results demonstrate that various forms of the MIP26K molecule undergo covalent modification during cataractogenesis of the diabetic rat lens, and that the degree of this change as monitored by binding of the anti-MIP26K peptide sera correlates with severity of the lens opacification.

Animals↗
PubMed1989

A covalent change in alpha crystallin during opacification of the Emory mouse lens.

Polyclonal antisera made against synthetic peptides corresponding to the N-terminus (anti-alpha NT), the C-terminus (anti-alpha CT), and to an internal sequence (anti-alpha 147-161) of bovine alpha-A2 crystallin have been used to quantitatively probe Western blots of proteins from the Emory mouse cataractous lens. Relative to proteins from transparent lenses of the control Carworth Farm Webster (CFW) mouse, there is no significant difference in binding of the anti-alpha NT and anti-alpha CT sera to alpha crystallin from lenses of cataractous Emory mice versus transparent lenses of CFW mice at any of the ages studied. In contrast, antiserum to an internal sequence (anti-alpha 147-161) binds significantly better to alpha crystallin from cataractous Emory mice lenses. Together, these results demonstrate covalent changes in the alpha crystallin molecule during opacification of the Emory mouse lens, which like those occurring in the human senile cataractous lens, result in increased binding of the anti-alpha 147-161 serum.

Aging↗
PubMed1989

Quantitation of high molecular weight protein aggregates in opaque and transparent parts from the same human cataractous lens.

Water soluble proteins from clear and opaque parts of the cortex of the same human cataractous lens were analyzed by high pressure liquid chromatography using a TSK 3000 SW gel filtration column. Studies of the 42 different microdissected parts from 7 different lenses demonstrated that the opaque parts possessed a higher percentage of high molecular weight material eluting with the void volume than did the corresponding clear part from the same lens. These results suggest that increased amounts of high molecular weight protein aggregates accompany the process of opacification in the cortex of the human cataractous lens.

Aged↗
PubMed1988

Covalent change in alpha crystallin during human senile cataractogenesis.

The high molecular weight aggregates (HMWA) obtained from normal and cataractous human lens nuclei have been resolved by SDS-polyacrylamide gel electrophoresis, and the alpha crystallin band has been probed with antisera made against the whole alpha crystallin molecule and with antisera made against synthetic peptides of alpha crystallin (alpha A2 147-161 and alpha A2 163-173). Quantitation of these antisera binding demonstrated that the anti-alpha A2 163-173 serum and the anti-alpha whole sera bound equally well to the alpha crystallin band from the HMWA fraction from normal and cataractous lenses. In contrast, the anti-alpha A2 147-161 serum bound little, if at all, to alpha crystallin from normal lenses, while it bound well to alpha crystallin from cataractous lenses. These results demonstrate a covalent alteration in the alpha crystallin molecule, and suggest a possible location of a covalent change that may occur during the cataractogenic process in the aged human lens.

Aged↗
PubMed1988

Changes in the major intrinsic polypeptide (MIP26K) during opacification of the Emory mouse lens.

Antisera against synthetic peptides corresponding to various regions of the Main Intrinsic Polypeptide (MIP26K) of fiber lens membranes have been used to probe Western blots of Emory mouse lens proteins resolved by SDS-polyacrylamide gel electrophresis. When compared with clear lenses from control animals of approximately the same age, the MIP26K component from Emory mouse lenses demonstrated no quantitative changes in the binding of anti-MIP26K256-263 and anti-MIP26Kwhole sera. In contrast, the MIP26K component from Emory mouse lenses bound significantly better to two other antisera directly against other parts of the molecule (antiMIP26K229-237 and anti-MIP26K252-259). Furthermore, this increase in binding was approximately proportional to the degree of lens opacification. Together, these results demonstrate that during the opacification process of the Emory mouse lens, there occur covalent changes in the MIP26K molecule that, in part, may mimic those occurring in the human senile cataract.

Aging↗
PubMed1988

High molecular weight aggregate from cataractous and normal human lenses: characterization by antisera to lens crystallins.

High molecular weight and low molecular weight fractions were obtained from total soluble proteins of human cataractous and normal lens nuclei. These fractions were analyzed using a solid phase radioimmunoassay that employed monospecific antisera to alpha, beta and gamma crystallins. Relative to the high molecular weight fraction from normal lens nuclei, the high molecular weight fraction from cataractous lens nuclei showed decreased binding to antisera specific for beta and gamma crystallins. These results demonstrate that the polypeptides of the high molecular weight fraction comprise a special class of polypeptides that have preferentially undergone covalent and/or structural changes during the process of human cataractogenesis.

Cataract↗