Characterization of novel sequences containing 3-O-sulfated glucosamine in glomerular basement membrane heparan sulfate and localization of sulfated disaccharides to a peripheral domain.

Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or L-iduronic acid (IdUA) and terminating in 2,5-anhydro[3H]mannitol (AnManH2), in addition to the nonsulfated component GlcUA beta 1----4AnManH2. Among these products two novel disaccharide units were identified as IdUA alpha 1----4AnManH2(3-SO4) and IdUA(2-SO4)alpha 1----4AnManH2(3-SO4); these accounted for 22% of the total sulfated species indicating that there are 2-3 residues of 3-O-sulfated glucosamine/heparan sulfate chain. The disulfated disaccharide was shown through its release by direct nitrous acid treatment to be situated in a GlcNSO3-IdUA(2-SO4)-GlcNSO3(3-SO4) sequence which is distinct from that in which 3-O-sulfated glucosamine is located in the antithrombin-binding region of heparins. Analyses of heparan sulfate from lens capsule, a nonvascular basement membrane, indicated the absence of sequences containing 3-O-sulfated glucosamine, although otherwise the sulfated disaccharides produced by hydrazine/nitrous acid/Na-B3H4 treatment (GlcUA beta 1----4AnManH2(6-SO4), IdUA alpha 1----4AnManH2(6-SO4), IdUA(2-SO4)alpha 1----4AnManH2 and IdUA(2-SO4)alpha 1----4AnManH2(6-SO4] were the same as from GBM. Examination of the GBM heparan sulfate domains after nitrous acid treatment indicated that the O- as well as N-sulfate groups are clustered in an iduronic acid-rich 10-disaccharide peripheral segment, while the internal region (approximately 20 disaccharides) is composed primarily of repeating GlcUA beta 1----4GlcNAc units. The localization of chain diversity to the outer region may facilitate interactions of the heparan sulfate with other macromolecular components.

Localization of insulin-like growth factor-1 binding sites in the embryonic chicken eye.

Insulin-like growth factor-1 (IGF-1) binding sites were localized in the embryonic chicken lens, retina, and retinal pigmented epithelium (RPE) with the use of autoradiography. Each of the ocular tissues exhibited specific binding of radiolabeled ligand. Labeling occurred over the entire surface of the lens from 6-day-old embryos, including the epithelium, basal ends of the fiber cells, and the annular pad. There was relatively little labeling over the lens nucleus. A similar pattern was seen in lenses from 19-day-old embryos. In isolated retinas from embryos of this age, the inner and outer plexiform layers were most heavily labeled. In the 19-day-old RPE, only the apical surface of the cells was heavily labeled. Electron microscopic studies revealed an apical layer of membranous material that may represent outer photoreceptor segments that remained attached to the RPE during dissection. It was uncertain, therefore, whether the IGF-1 was binding to sites on the RPE or to these membrane fragments.

Effects of long-term diabetes and galactosaemia upon lens and retinal mRNA levels in the rat.

The levels of mRNAs encoding the alpha 1 chain of collagen IV and the B1 chain of laminin were assayed in the lenses and retinas of long-term (28-week) diabetic and galactosaemic rats in order to gain some insight into the effects on basement membrane (BM) synthesis in these tissues. mRNAs coding for beta-actin, glucose transporter protein and the alpha 2 catalytic subunit of Na+,K(+)-ATPase were also assayed to determine whether any effects on BM-coding mRNA levels were specific. Long-term diabetes had no significant effect on the levels of alpha 1 (IV) collagen mRNA but caused a significant reduction in the laminin B1 message in the lens. In the same samples, the level of the glucose transporter protein mRNA was found to be elevated significantly in the diabetic tissue, whereas the mRNAsen coding beta-actin and alpha 2 Na+,K(+)-ATPase were unaffected in comparison with age-matched controls. Long-term galactosaemia resulted in significant increases in the levels of all mRNAs assayed when expressed per micrograms total RNA used for each analysis. However, this effect appeared to be due to a specific loss of ribosomal RNA from these severely cataractous lenses. When related to the beta-actin mRNA internal control, the levels of mRNA in the galactosaemic lenses were very similar to that found in the diabetics. Laminin B1 mRNA levels were decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of tryptic peptides from the C-terminal region of alpha-crystallin from cataractous and normal human lenses.

Antisera have been made to synthetic peptides corresponding to the expected tryptic fragments from the C-terminal region of human alpha A2 crystallin (T19 corresponds to residues 158-163; T20 corresponds to residues 164-173). These antisera were used on conjunction with a sensitive radioimmunoassay, to identify the elution times of peptides resolved on a C18 column from a tryptic digest of water soluble and water insoluble proteins from the human lens. Isolation and purification of the peptides reactive with the anti-peptide sera, followed by the use of tandem mass spectrometry to determine the amino acid sequences in the peptides, demonstrated that the antisera reacted specifically with the T19 and T20 sequences. Using the antisera specific for the T19 sequence, analysis of the peptides resolved from tryptic digests of individual lenses demonstrated no major differences between the elution profiles of five normal vs. ten cataractous lenses, while analysis of the same digests with the antiserum to the T20 sequence demonstrated major changes in reactivity and/or elution time of tryptic peptides from eight of the cataractous lenses analyzed. Together, these studies strongly suggest that during human cataract formation, covalent changes occur in the C-terminal region of the alpha A2 molecule. In addition, these studies provide the general methodology, whereby antisera specific for known sequences of a polypeptide chain, can be used to locate the sequences involved in covalent modification during the process of senile cataractogenesis of the human lens.

Calcium-induced aggregation of the urea-deaggregated human lens insoluble protein.

Calcium-induced aggregation of the urea-deaggregated insoluble protein (US-fraction) derived from human lens was investigated by use of a simple light scattering method. The initial rate of aggregation was shown to increase with progress of senescence of the lens, and to be much higher in the nucleus as compared to the cortex. The aggregation was found to be more active in the US-fraction from cataractous lens than in that of normal lens at same age. A calcium sensitive polypeptide (CSP) was isolated from the US-fraction from cataractous human lenses by gel filtration followed by DEAE-cellulose column chromatography in the presence of 7 M urea. The molecular size of the CSP in the dissociated state was assessed to be 4.2 kDa by urea-SDS-PAGE, and the size in the associated state to be 450 kDa by thin layer gel filtration. With addition of calcium ion to the latter, it aggregates rapidly to form much larger aggregates and eventually precipitates out. The aggregation rate of the CSP is shown to increase exponentially with an increase in the concentration of both CSP and calcium ion, indicating the involvement of complicated mechanisms in the aggregation process. Effects of divalent ions other than calcium ion on the aggregation process were also examined. It is revealed that the effect of manganese ion is little less than that of calcium ion, whereas magnesium and strontium ions are much less effective as compared to the former two ions.

Partial amino acid sequence of the major intrinsic protein (MIP) of the chicken lens deduced from the nucleotide sequence of a cDNA clone.

A cDNA clone of the major intrinsic protein (MIP) of the chicken lens was isolated. This clone covers the C-terminal half of the coding region and 3'-untranslated region including a polyadenylation signal. Comparison with the bovine MIP cDNA sequence revealed that: (1) the amphilphilic transmembrane helix in bovine MIP is highly hydrophobic in chicken MIP, and is thus unlikely to offer a hydrophilic lining of the transmembrane pore, and (2) the possible calmodulin binding site is conserved especially at amino acid residues which are postulated to be important in its binding with calmodulin. Northern blotting revealed the presence of transcripts of different lengths, two of which correspond closely to the transcripts of bovine MIP.

[Detection of sorbitol content in crystalline lens of normal rats and rats with diabetic cataract by 1H-NMR].

The pulse Fourier NMR was employed to measure the artificial diabetic cataract lens at various stages of its formation, and the lenses of the normal rats. Data obtained by using this method show that all the peaks that of water concentrate in the range of delta less than 4 ppm. The peak value at delta = 3.20 ppm is on a marked increase during the formation of cataract which is caused by the phosphate metabolites, such as GPC, ATP, ... etc, in cataract lens. With the development of the disease, the peak width at delta = 3.73 ppm becomes greater and greater, which shows that the activity of sorbitol dehydrogenase has decreased. This leads to a high concentration of the sorbitol in the cataract lens. Consequently, the osmosis pressure in the cataract lens is increased, and excessive water might dip into the crystalline lens to keep the balance of the osmosis pressure. And this might result in the hydration of the fiber cell of the crystalline lens, which might cause a swelling or blisters. These results are in favour of the prolongation of the relaxation time of cataractous lens reported in our other papers, and also support those gained by biochemical studies issued in the medical literature.

[Study on change in zinc concentration for diabetic cataractous lenses in rats by means of XIXA].

The SD rats were injected with streptozotocin to induce diabetic cataract. Element Zn as well as K, Ca, Mn, Fe, Cu, Pb, Se, etc. in the eye lenses was determined by means of X-ray induced X-fluorescence analysis (XIXA). Compared to values for normal healthy rats, the concentration of Zn and Fe in the cataractous lenses increased, the concentration of K decreased, and that of the others did not change prominently. The change in Zn concentration was probably to meet the needs of the metabolic adjustment in the eye lenses, which would not be the reason for directly inducing diabetic cataract.

Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation.

Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with 32P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.

[Study on the variation of relaxation time in diabetic cataractous lenses of rats by using NMRA].

Nuclear magnetic resonance analysis (NMRA) was used to measure the relaxation time T1 and T2 of the protons in the different-stage-diseased lenses of rats with diabetic cataract caused by the streptozotocin, and the results were put in comparison with those obtained from the control group, showing that after one month of medication the values of T1 and T2 in the cataractous lenses are greater than those gained from the control group. The variation, as time goes on, becomes greater and greater. T1 and T2 indicate the speed of the free induction decay (FID) signals, and the variation of the FID signals are in direct proportion to that of the proton content of the samples. This shows that an increase in T1 and T2 indicates an increase in the water content in the diseased lenses, and the changes in ratio of "bound" water to free water. Such changes may be explained that the space for "bound" water is occupied by some other macromolecules, causing the alteration of the macromolecular structure, and consequently the aggregation of protein and the degeneration or dehydration or dehydration of the membrane occur, which may give rise to another scattering centre. So the scattering of the lens increases and its transparency decreases. This result is in agreement with some reports in the medical literature. As T1 and T2 are quite different in the normal and pathological tissues, and the protons are the most extensive nucleus in all biological tissues, NMR is a very important method to determine the proton relaxation time in the biological tissue and to judge if it is normal or not.(ABSTRACT TRUNCATED AT 250 WORDS)

Identity of the calmodulin-binding proteins in bovine lens plasma membranes.

Two bovine lens plasma membrane proteins have been identified that bind calmodulin in a Ca2(+)-dependent fashion. Electrophoretic analysis of lens membrane proteins photoaffinity-labeled with benzophenone[125I]calmodulin confirmed our previous observation [Louis, Johnson and Turnquist (1985) Eur. J. Biochem. 150, 271-8] that two major products are formed with Mr = 46 kDa and 36 kDa. Limited proteolysis of lens membrane proteins with chymotrypsin resulted in the formation of a 21-kDa-fragment that was derived from the hydrolysis of the major lens membrane protein MP26; this correlated with the loss of the 46-kDa complex, and the formation of a 41-kDa photoaffinity-labeled complex. Use of the [125I]calmodulin gel overlay procedure confirmed that the 46-kDa and 41-kDa photoaffinity-labeled complexes reflect the interaction of calmodulin with both MP26 and its 21-kDa chymotryptic fragment, respectively. Proteolysis of lens membranes with higher concentrations of chymotrypsin resulted in the hydrolysis of the 18-kDa protein which correlated with the generation of a 12-kDa fragment; this paralleled the loss of the 36-kDa photoaffinity labeled complex and the formation of a 28-kDa-complex. The [125I]calmodulin gel overlay procedure demonstrated that the 36-kDa and 28-kDa photoaffinity-labeled complexes reflect the interaction of calmodulin with the 18-kDa lens membrane protein and its likely 12-kDa chymotryptic fragment, respectively. Identification of these calmodulin-binding proteins suggests that MP26 and the 18-kDa membrane protein are likely candidates for the proposed calcium sensitive, calmodulin-dependent gating of lens fiber cell junctions.

Hydrodynamic characterization of the major intrinsic protein from the bovine lens fiber membranes. Extraction in n-octyl-beta-D-glucopyranoside and evidence for a tetrameric structure.

The major protein from the bovine lens fiber cell membranes, the 26-kilodalton protein (major intrinsic protein (MIP26)), has been solubilized in n-octyl-beta-D-glucopyranoside and purified by gel filtration. The final preparation was free of detergent micelles. Gel electrophoresis in denaturing conditions has confirmed the purity of the protein sample. A s20,w of 5.55 S, obtained from analytical ultracentrifugation, and a D20,w of 3.62 x 10(-7) cm2 s-1, obtained from photon correlation spectroscopy, resulted in a molar mass of (176,000 +/- 15,000) g/mol for the protein-detergent complex using the Svedberg relation. The measured detergent content of 0.71 g of detergent/g of protein resulted in a calculated partial specific volume of 0.787 cm3/g for the protein-detergent complex and a molar mass of 103,000 g/mol for the protein moiety. This allowed us to conclude that the protein-detergent complex contains four copies of the MIP26 protein, which supports the suggestion that in vivo the MIP26 molecules cluster in tetramers to form a pore-like structure.

[Analysis of trace elements in cataractous lenses and environmental samples in Tibet].

Trace elements of Cu, Fe, Zn, Mg, and Se in Tibetan cataractous lenses and also in local samples of earth, drinking water, and staple foods were analyzed to estimate the possible relationship between environmental factors and primary cataract formation in Tibet. The Fe, Mg and Se contents in Tibetan cataracts were significantly lower in comparison with levels in those of Chengdu area, particularly the Se contents were low in both cataracts and local environment, suggesting a probable correlation between cataract formation and low Se contents in food and water.

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.

Comparison of membrane phospholipids of the rabbit and pig crystalline lens.

Crystalline lenses excised from 5-7-month-old rabbits and pigs were extracted for lipids with chloroform/methanol, 2/1 using the Folch method. The extracted crude lipids were analyzed at 202 MHz by 31P-NMR spectroscopy. Twelve membrane phospholipids were detected. Both rabbit and pig phospholipid profiles contained phosphatidylcholine (PC), lysophosphatidylcholine, phosphatidylcholine plasmalogen, phosphatidylethanolamine (PE), phosphatidylethanolamine plasmalogen (PE plas), phosphatidylserine, sphingomyelin (SPH), and an uncharacterized phospholipid. In addition, pig lens profiles contained lysophosphatidylethanolamine, phosphatidylinositol, cardiolipin and phosphatidylglycerol. The data indicate that these two animal models have significant differences in membrane phospholipid profiles. In each species, however, the bulk phospholipid component resides in the neutral phospholipids PC, PE, PE plas, and SPH.

31P NMR of phospholipid glycerol phosphodiester residues.

Saponification of extracted tissue phospholipids yields a set of isolated glycerol 3-phosphoryl phospholipid polar headgroups from which semi-quantitative 31P NMR spectra can be obtained. The resonance signals from these molecules, which frequently have been reported as uncharacterized phosphate signals observed in perchloric acid extracts of tissue, can be used as an aid in the characterization of isolated phospholipids and of tissue phospholipid 31P NMR profiles. 31P NMR chemical-shift values of the resonances at pH 7 in water and relative to 85% phosphoric acid are: glycerol 3-phosphocholine (-0.13 delta), glycerol 3-phosphoethanolamine (0.42 delta), glycerol 3-phospho(monomethyl)ethanolamine (0.29 delta), glycerol 3-phospho(dimethyl)ethanolamine (0.16 delta), glycerol 3-phosphoserine (0.14 delta), glycerol 3-phosphoinositol (-0.07 delta), glycerol 3-phosphoglycerol (0.92 delta), bis(glycerol 3-phospho)glycerol (0.79 delta), serine ethanolamine phosphodiester (-0.46 delta), glycerol 3-phosphate (0.60 delta; 4.29 delta at pH 10) glycerol 2-phosphate (0.15 delta; 3.92 delta at pH 10). In addition, analysis of extracted cancer tissue phospholipid samples yielded a new and uncharacterized polar headgroup fragment with a chemical-shift value of 0.29 delta that is independent of sample pH.

Identification of the 32 kDa components of bovine lens EDTA-extractable protein as endonexins I and II.

EDTA-extractable protein (EEP) is a mixture of major lens membrane proteins with molecular masses ranging from 32 kDa to 40 kDa. These bind to the lens membrane in a Ca2(+)-dependent manner. In the present study we have identified and purified two distinct 32 kDa components of EEP (designated as EEP 32-1 and EEP 32-2) from bovine lens that inhibit phospholipase A2 activity. Both EEP 32-1 and EEP 32-2 bind to phospholipid-containing liposomes and actin filaments in a Ca2(+)-dependent fashion. Immunochemical studies and two-dimensional electrophoreses demonstrate that the two proteins are distinct from one another. Both EEP 32-1 and EEP 32-2 are clearly different from calpactin (lipocortin) or its proteolytic fragments because they did not react with anti-[human placenta calpactin (lipocortin)] antibody. Our results also indicate that EEP 32-1 is very similar to endonexin I and that EEP 32-2 corresponds to endonexin II.

Quantitative photoacoustic spectroscopy of cataractous human lenses.

Quantitative photoacoustic spectra of the nuclei of cataractous human lenses with various degrees of colouration and opacification were measured in the spectral range 250-600 nm. The lens nuclei were obtained from 20 cataractous patients through extracapsular cataract extraction. These measurements yield the light loss per unit path length in the nucleus of cataractous lenses.

N-cadherin detected in the membrane fraction of lens fiber cells.

N-cadherin was identified as a glycoprotein present in the fiber cell membranes of frog, chick, bovine, rabbit and human lenses. The molecular size of N-cadherin varies with the species. Homogenization of the chick lens in the presence of Ca2+ resulted in a decrease in the concentration of N-cadherin. This suggests that the lens contains a Ca2(+)-activated protease which can act on N-cadherin.

Micro-quantitation of lens myo-inositol by anion exchange chromatography.

A method capable of the determination of pmole quantities of myo-inositol in mgr amounts of tissue by anion exchange chromatography is detailed for use in lens and potentially other tissues. Samples were rendered protein-free through ZnSO4/Ba(OH)2 precipitation, lyophilized and reconstituted in water just prior to analysis. An aliquot of sample was injected onto an anion exchange column and eluted with a 0.045 M sodium hydroxide mobile phase. Each analysis requires 30 minutes to complete. Average recovery of myoinositol added to lens sample prior to injection was 100.6%. The coefficient of variation for repeated sample injections was 2.9%. Rabbit lens averaged 11.4 mumol/gr wet weight with epithelium containing 8.5 mumol/gr wet weight while human lens contained 20.1 mumol/gr wet weight and human epithelial cells had 17.5 nmol/mgr protein.