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L Takemoto

Publications and source records attributed to L Takemoto.

68 records · Page 4Linked to original sources
PubMed1988

Characterization of disulfide-linked crystallins associated with human cataractous lens membranes.

In order to characterize possible disulfide-linked interactions between lens fiber cell membranes and crystallins, two-dimensional diagonal electrophoresis has been used in combination with Western blot analysis. When these blots were probed with monospecific antisera against alpha, beta and gamma crystallins, membrane from five individual normal lenses showed no disulfide-bonded components. Membrane from 13 individual cataractous human lenses showed no disulfide-bonded alpha crystallin, but did show significant amounts of disulfide-bonded beta crystallin in four out of the 13 lenses studied, and significant amounts of disulfide-bonded gamma crystallin in 10 out of the 13 lenses studied. Together, these studies demonstrate that intermolecular disulfide bonding of crystallins to purified fiber cell membranes is found only in cataractous lenses, and that the predominant polypeptide species involved in this interaction is gamma crystallin.

Cataract↗
PubMed1987

Major intrinsic polypeptide (MIP26K) of the lens membrane: covalent change in an internal sequence during human senile cataractogenesis.

Polyclonal antisera to three synthetic peptides of bovine MIP26K have been used in combination with Western blot analysis to probe for changes of the MIP26K molecule during human senile cataractogenesis. Anti-MIP26K229-237 binds well to the 26K component from cataractous lens membranes, but binds poorly to the same component from normal lens. In contrast, antisera to two other sequences of MIP26K (anti-MIP26K252-259 and anti-MIP26K256-263) bind approximately equally well to the 26K component from either cataractous or normal lens. Together, these results demonstrate that during cataract development there is a selective covalent change in a region of the MIP26K molecule that may have profound effects upon the ability of this molecule to facilitate intercellular communication between lens fiber cells.

Amino Acid Sequence↗
PubMed1987

Cleavage from the N-terminal region of beta Bp crystallin during aging of the human lens.

Polyclonal antisera have been made to synthetic peptides that correspond to the N-terminal (residues 1-12) and C-terminal (residues 195-204) sequences of bovine beta Bp crystallin. Both anti-beta Bp1-12 and anti-beta Bp195-204 recognize specifically the beta Bp component of bovine lens. In the young human lens, anti-beta Bp195-204 recognizes predominantly the 26,000 MW form of beta Bp, while in older lenses this same antiserum recognizes mainly the 22,000 MW in vivo proteolysis product. In contrast, during aging of the normal human lens anti-beta Bp1-12 recognizes only decreasing amounts of the 26,000 MW form of beta Bp, with no binding to the 22,000 MW form of this polypeptide. These results suggest that during aging of the normal human lens, the N-terminus of beta Bp is the preferred site of in vivo proteolysis.

Adolescent↗
PubMed1987

Covalent changes at the N- and C-terminal regions of gamma crystallin during aging of the normal human lens.

Polyclonal antisera have been made to synthetic peptides corresponding to the N- and C-terminal regions of the putative gamma 1-2 gene from human lens. These antisera are specific for gamma crystallin, showing no cross-reactivity with any polypeptides of the alpha- and beta-crystallin fractions. Western blot analysis demonstrates a dramatic decrease in the binding of these antisera to gamma crystallin during aging of the normal human lens, while identical analysis with polyclonal antisera to the major cyanogen bromide fragment (19,000 MW) of human gamma crystallin shows little, if any, change during aging. Together, these demonstrate that antisera to synthetic peptides of the N- and C-terminal regions of the gamma 1-2 gene are very specific probes that can demonstrate extensive covalent modification from both ends of the gamma crystallin molecule during aging of the normal human lens.

Adolescent↗
PubMed1987

Quantitation of membrane-associated crystallins from aging and cataractous human lenses.

Membrane from individual cataractous and normal human lenses has been prepared by discontinuous sucrose gradient centrifugation. A sensitive solid-phase radioimmunoassay has been developed to quantitate the levels of alpha, beta, and gamma crystallins associated with membrane obtained from each sucrose gradient interface. The results of this analysis demonstrate significant levels of all three crystallins associated with purified membrane. Although the relative percentage of each membrane-associated crystallin does not change during aging and/or cataractogenesis of the human lens, membrane from cataractous lenses in characterized by greater total amounts of crystallins associated with purified membrane obtained from denser interfaces of the discontinuous sucrose gradient.

Adolescent↗
PubMed1987

Comparison of microdissected sections from the human cataractous lens by antisera to synthetic peptides.

Polyclonal antiserum has been made against beta crystallin from human lens, and against synthetic peptides corresponding to the N- and C-terminal sequences of bovine beta Bp crystallin. A solid-phase radioimmunoassay has been used to quantitate binding of these antisera to soluble proteins from microdissected sections. The results of this analysis demonstrate the feasibility of using radioimmunoassay analysis in combination with peptide antisera to determine statistically significant changes in protein antigenicity from opaque versus transparent regions from the same human cataractous lens.

Cataract↗
PubMed1986

Covalent change of major intrinsic polypeptide (MIP26K) of lens membrane during human senile cataractogenesis.

Polyclonal antisera have been made against synthetic peptides corresponding to the C-terminal octapeptide and N-terminal nonapeptide of bovine MIP26K. Western blot analysis demonstrated significant binding of the C-terminal antiserum to MIP26K of both normal and cataractous human lens. In contrast, the N-terminal antiserum bound to MIP26K of normal lenses, but failed to bind to MIP26K of 7 out of 10 cataractous lenses studied. These results demonstrate for the first time, a covalent change in MIP26K during human cataractogenesis, and strongly suggest that the location of this change is in the N-terminal region of the polypeptide.

Aged↗
PubMed1986

Major intrinsic polypeptide (MIP26K) from human lens membrane: characterization of low-molecular-weight forms in the aging human lens.

Antisera made against a C-terminal octapeptide of bovine MIP26K and against an N-terminal nonapeptide from the same protein have been used to determine the location of age-dependent cleavage sites of MIP26K in the human lens. Neither the C-terminal antiserum (anti-MIP26K256-263) nor the N-terminal antiserum (anti-MIP26K1-9) binds to the 22,000 MW form of MIP26K, suggesting cleavage from both the N- and C-terminus during lens aging. Anti-MIP26K256-263, but not anti-MIP26K1-9, binds to 20,000 and 15,000 MW forms of MIP26K, demonstrating that age-dependent production of these forms occurs by cleavage from the N-terminal side of the molecule. Together, these results show that age-dependent processing of MIP26K in the human lens occurs from both ends of the molecule, with cleavage from the N-terminal end being mainly responsible for production of the lower-molecular-weight 20,000 and 15,000 MW components.

Adolescent↗
PubMed1986

Covalent changes in MIP26K during aging of the human lens membrane.

Rabbit antisera have been made to the C-terminal octapeptide of bovine MIP26K (residues 256-263) and to an octapeptide near the C-terminus (residues 252-259). Use of these antisera in Western blot analysis of membrane from young vs old human lenses, and from cortex vs nucleus of old human lenses, suggest age-related covalent changes in the C-terminus of MIP26K. These age-related changes in anti-256-263 and anti-252-259 sera can be mimicked by treatment of lens membrane with carboxypeptidase Y, suggesting the loss of 1-8 amino acid residues from the C-terminus of the 26,000 dalton form of MIP26K during aging of the normal human lens.

Aging↗
PubMed1985

Major intrinsic polypeptide (MIP26K) from lens membrane: reconstitution into vesicles and inhibition of channel forming activity by peptide antiserum.

Bovine and human lens membrane, when reconstituted into lipid vesicles containing oxidized cytochrome C, will mediate the transmembrane passage of ascorbate into the vesicles, where the reduction of cytochrome C is measured spectrophotometrically. This channel forming activity is specifically inhibited by antiserum made against a synthetic octapeptide near the C-terminus of MIP26K. Together, these studies describe a direct and more sensitive assay system for measurement of channel-forming activity of MIP26K, and suggest that the C-terminus of this molecule may be particularly important in the regulation of channel formation.

Animals↗
PubMedDate not supplied

The possible role of alpha-crystallins in human senile cataractogenesis.

alpha-Crystallins possess molecular chaperone properties and are one of the most abundant of the lenticular proteins. Posttranslational modifications of these proteins have been implicated as a possible etiology of human cataracts. This article will review current knowledge concerning the effects of known posttranslational modifications upon the molecular chaperone properties and aggregation behavior of alpha-A and alpha-B crystallin. Based upon these effects, experimental approaches will be discussed that may be useful in the development of reagents that may selectively inhibit the cataractogenic process in the aging human lens.

Aged↗