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L J Takemoto

Publications and source records attributed to L J Takemoto.

At least 55 records · Page 3Linked to original sources
PubMed1985

Reconstitution of MIP26 from single human lenses into artificial membranes. I. Differences in pH sensitivity of cataractous vs. normal human lens fiber cell proteins.

Reconstitution of the lens fiber cell protein known as MIP26 into liposomes composed of heterologous phospholipids was achieved; this protein renders the liposomes permeable to low molecular weight compounds. MIP26 from either bovine or human lenses was capable of forming channels in artificial membranes. The assay technique was sufficiently sensitive to allow reconstitution of MIP26 from single human lenses, enabling us to examine the function of channels from either cataractous or age-matched normal lenses. Decreases in pH can cause these channels to close, analogous to the hypothesized channel closing in the in vivo situation. The pH optimum of reconstituted channels in liposomes containing MIP26 from bovine lenses or normal human lenses is very sharp; but is substantially broadened if the liposomes contain MIP26 from cataractous human lenses. This latter result suggests a functional alteration in human lens membranes which is correlated with the development of human senile cataract.

Animals↗
PubMed1985

Partial characterization of three distinct populations of human gamma-crystallins.

Certain low molecular weight crystallins may be involved preferentially in the process of human cataractogenesis. Three distinct populations of monomeric crystallins with molecular weights ranging from 19,000 to 24,000 have been demonstrated previously by electrophoresis in the presence of sodium dodecyl sulfate (SDS). The authors now report the chromatographic separation of these three components from young human lenses. Each of the three species has unique profiles on isoelectric focusing and native polyacrylamide gels as well as on SDS gels. All three species show similar patterns of age-related modification in their charge characteristics. These three populations of polypeptides all appear to be gamma-crystallins. The relative amounts of the three vary with age, which accounts for the greater ease of their separation in extracts from young lenses as compared with adult lenses.

Aged↗
PubMed1984

Leucine aminopeptidase from bovine lens and hog kidney. Comparison using immunological techniques, electron microscopy, and X-ray diffraction.

The crystallization of leucine aminopeptidase from hog kidney is reported for the first time. The crystals which diffract to 4-A resolution have the space group P2(1)2(1)2(1) (a = 186.3 A, b = 223.2 A, and c = 80.5 A) and contain four hexamers per unit cell, or one per asymmetric unit. Electron micrographic images of hog kidney leucine aminopeptidase are indistinguishable from micrographs of beef leucine aminopeptidase taken under the same conditions (10). These reveal an equilateral triangle of about 85 A per side, seemingly made of three 40-A diameter spheres. This triangle is circumscribed by another concentric, less-dense triangle of 120 A per side which is rotated 60 degrees with respect to the inner triangle. Immunodiffusion and microcomplement fixation assays indicate that the two enzymes share greater than 90% amino acid sequence homology. This similarity is corroborated by peptide maps of tryptic fragments of the radioiodinated enzymes. The model of the quaternary structure proposed to explain the appearance of electron micrographs of single molecule and crystalline bovine lens enzyme also describes the hog kidney enzyme equally well. That the model of leucine aminopeptidase originally proposed for the beef enzyme also can be used to describe hog kidney leucine aminopeptidase crystal packing in the highly anisometric unit cell provides further corroboration that leucine aminopeptidase in these two species is a hexamer based on two trimers each made of three bilobal promoters.

Animals↗
PubMed1984

High molecular weight aggregates from human cataracts: characterization by Western blot analysis.

The formation of covalently linked, high molecular weight protein aggregates has been thought to play an important role in opacification of the human lens. Antisera were used in Western blot analysis to demonstrate the involvement of all major classes of lens proteins (alpha, beta and gamma crystallin; the major intrinsic membrane polypeptide) in covalent aggregation. Of these classes, aggregation of gamma and beta crystallins via intermolecular disulfide bonding and aggregation of the major intrinsic membrane polypeptide via intermolecular, non-disulfide bonding were more pronounced in cataractous as compared with normal lenses.

Cataract↗
PubMed1984

Cyclic GMP phosphodiesterase from bovine retina. Evidence for interspecies conservation.

A light-activated phosphodiesterase (PDE) from retinal rod outer segments (ROS) has been strongly implicated as a possible mediator in the propagation of the visual response. In view of the probable importance of this enzyme in the visual system, a comparison of the PDE proteins from ROS of different evolutionary species was made. Partial purification of the PDE, as measured by enzymic activity, followed by resolution of the protein components on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicated that the major Coomassie Blue-staining species in the ROS of all species studied is a doublet of 84000-88000 Da. After radioiodination, this doublet was converted in all but the frog proteins into a single band of 85000 Da. Two-dimensional tryptic peptide mapping of the radioiodinated peptides indicated that at least six major peptides of the putative PDE have been conserved in all of the species studies. If this protein is indeed associated with PDE activity, such conserved peptides may play an important role in the catalytic and/or regulatory functions of the enzyme.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1984

Clinicobiochemical correlations in aging-related human cataract. The Pan American Association and American Journal of Ophthalmology lecture.

In a series of 50 consecutive adult patients (24 men and 26 women) we studied aging-related cataract by means of comprehensive presurgical examination of the patient, the eyes, and the cataractous lens; cataract sample collection from predetermined sites during surgery; biochemical analyses with high-performance liquid chromatography of each individual lens sample; and computer-assisted biostatistical data analysis. Compared with normal values established on clear human lenses obtained from a donor eye program, the ratios of 23,000 (23K) crystallin to total beta, gamma, and low molecular weight soluble proteins were statistically significantly lower in the anterior axial lens cortex (P less than .001) and in the lens nucleus (P less than .001). Increased grades of cortex opacification and nucleus opacification were generally associated with progressively decreased 23K crystallin ratios. In this series of patients with diverse cataract-associated systemic diseases, family histories, ophthalmic disorders, and drug-use patterns, decreased 23K crystallin ratios were a sensitive marker for aging-related cataract. Similar biochemical abnormalities in patients with multiple cataract-associated factors may have important clinical implications.

Adult↗
PubMed1983

Major intrinsic polypeptide of lens membrane. Biochemical and immunological characterization of the major cyanogen bromide fragment.

A protein of Mr 26000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the Mr 26000 polypeptide from bovine lenses yields a major fragment of Mr 15000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the Mr 26000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the Mr 26000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the Mr 26000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the Mr 26000 polypeptide is buried within the lipid bilayer.

Amino Acid Sequence↗
PubMed1983

Presence of low molecular weight polypeptides in human brunescent cataracts.

Microdissected sections from opaque & brunescent lens nuclei contain low molecular weight (4,000-8,000 dalton) polypeptides not found in microdissected sections of transparent lens nuclei. Tryptic digestion of these polypeptides from different cataracts reveal similar peptide maps. Together, these results support the involvement of proteolysis in human cataractogenesis, and suggest the possibility of similar molecular mechanisms occurring in cleavage of lens polypeptides during formation of the opaque & brunescent human cataract.

Aged↗
PubMed1983

Differences between liver gap junction protein and lens MIP 26 from rat: implications for tissue specificity of gap junctions.

Liver gap junctions and gap-junction-like structures from eye lenses are each comprised of a single major protein (Mr 28,000 and 26,000, respectively). These proteins display different two-dimensional peptide fingerprints, distinct amino acid compositions, nonhomologous N-terminal amino acid sequences and different sensitivities to proteases when part of the intact junction. However, the junctional protein of each tissue is well conserved between species, as demonstrated previously for lens and now for liver in several mammalian species. The possiblity of tissue-specific gap junction proteins is discussed in the light of data suggesting that rat heart gap junctions are comprised of yet a third protein.

Amino Acid Sequence↗
PubMed1983

Analysis of low molecular weight fractions in human senile cataractous lens.

Previous studies have suggested that changes occur in low molecular weight proteins during the progression of human senile cataract. In this report we have employed the reverse phase mode of high performance liquid chromatography (HPLC) and characterized the patterns of changes in the fractions of low molecular weight substances. Two different types of cataractous lenses, brown and yellow types, were used. Classification of cataract stages in each type was determined by measuring the Na+/K+ ratio in each lens. The profile of low molecular weight substances loosely bound to the reverse phase column changed significantly during cataract progression. In the brown type cataractous lenses, however, the low molecular weight substances changed after the middle stage of cataract progression, while in the yellow type cataractous lenses, the changes started during the early stages of cataract development. These experiments demonstrated that the reverse phase mode of HPLC is a sensitive and specific method for characterization of the quantitative differences in low molecular weight compounds during the progression of human senile cataract.

Aged↗
PubMed1982

Peptide mapping of multiple forms of cyclic nucleotide phosphodiesterase.

Purified multiple forms of 3':5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) were analyzed using two-dimensional tryptic pep]tide mapping of radioiodinated peptides. Comparisons of peptide maps of rat liver insulin-sensitive phosphodiesterase (PDE) with rat brain calmodulin-sensitive PDE suggest that some peptides co-migrate (31-43% co-migration). However, except for a single peptide, bovine retinal rod outer segment PDE, peptide maps appear unrelated to the other two forms (7-12% co-migration). In contrast, peptide maps of a 36,000-dalton proteolysis product of calmodulin-sensitive PDE are highly related to the peptide maps of a rat brain calmodulin-sensitive holoenzyme (73% co-migration). These results suggest that the multiple PDE forms are distinct molecular entities.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Identification of tumor-associated antigens on ultraviolet light-induced tumors using antitumor antibodies developed in ascites fluid.

A method is described which leads to the production of large amounts of ascites containing antitumor antibody in small numbers of mice. The antibody was then used to identify and characterize tumor-associated antigens on an ultraviolet light-induced murine skin fibrosarcoma. The antibody showed specific complement-dependent cytotoxicity to the homologous tumor and to an allogeneic tumor line which displayed a glycoprotein viral determinant with a molecular weight of 70,000 on its surface. Absorption of the immune ascites with other tumor cell lines removed the cytotoxicity in relation to the presence of the glycoprotein. Isolation of the tumor cell surface components binding antibody revealed two components with molecular weights of approximately 70,000 and 60,000. The Mr 70,000 component was identified as viral gp70 by peptide mapping.

Animals↗
PubMed1982

Intermolecular disulfide bonding of lens membrane proteins during human cataractogenesis.

Two-dimensional diagonal electrophoresis has been used to characterize intermolecular disulfide bonding of membrane proteins from lenses of cataractous and normal patients. A component of approximately 18,000 daltons, linked via intermolecular disulfide bonding, was found in membrane preparations from 10 of 17 cataracts studied. In comparison, membrane from only 1 of 12 normal lenses of approximately the same age range was found to contain intermolecular disulfide bonding of a component of similar molecular weight. Treatment of normal lens with the oxidizing agents cupric sulfate and 1,10-phenanthroline resulted in intermolecular disulfide bonding of the 18K component in a manner similar to that found in cataractous lenses. Together these results demonstrate that human cataractogenesis is many times accompanied by intermolecular disulfide bonding of a membrane component of 18K and suggest that this intermolecular bonding may be the result of the previously reported oxidative insult of the lens during human cataract formation.

Adult↗