C.R. Fleschner

Connexin 46 and connexin 50 in selenite cataract.

The purpose of this work was to determine if the lens gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) were altered with the development of selenite-induced cataract. Cataracts were induced in young Sprague-Dawley rats with a single subcutaneous injection of sodium selenite; age-matched uninjected rats served as controls. Membrane fractions were isolated from homogenates of cortex and nucleus of normal and cataractous lenses by differential and discontinuous sucrose gradient centrifugation. Aliquots of urea-insoluble protein from membrane fractions were analyzed by quantitative densitometry of Western blots probed with antibodies to Cx46 and Cx50. A significant decrease in the more slowly migrating Cx46-reactive band, which represents phosphorylated Cx46, was found in the major membrane fraction of the cortex of cataractous lenses. There was no significant difference in the amounts of either Cx46 or Cx50 associated with selenite cataract in any of the membrane fractions examined. These results suggest that alteration of gap junction function (as evidenced by the change in phosphorylation of Cx46) may be associated with the development of the selenite cataract, but that neither Cx46 nor Cx50 is subject to the well-characterized proteolysis associated with the selenite cataract model.

Intermediate filament cytoskeletal proteins associated with bovine lens native membrane fractions

PURPOSE To examine the intermediate filament cytoskeletal proteins associated with native membrane fractions isolated from bovine lenses. METHODS Decapsulated bovine lenses were divided into cortex and nucleus. The lens regions were homogenized and separated into water-soluble and water-insoluble fractions by centrifugation. Sedimenting membrane fractions were isolated from the water-insoluble fraction by discontinuous sucrose-density-gradient centrifugation and the non-sedimenting membrane fractions were isolated from the Kbr high-density water-soluble fractions by flotation, during overnight centrifugation. The intermediate filament peptides of the membrane fractions were examined by Western blot analysis, using monoclonal antibodies to filensin, cytoskeletal protein 49 (CP49) and vimentin. RESULTS Filensin immunoreactive peptides were found in all membrane fractions of both cortex and nucleus. The parent 115 kDa filensin was found almost exclusively in the urea-soluble protein of cortical membrane fractions, and was the predominant filensin immunoreactive peptide only in the urea-soluble protein of the cortical sedimenting membrane fraction isolated from the 25%/45% sucrose density interface. The predominant filensin immunoreactive peptide of all other samples migrated with a M(r) of 53 kDa. CP49 immunoreactive peptides were found almost exclusively in the urea-soluble protein of all membrane fractions from both the cortex and nucleus. The cortical non-sedimenting membrane fraction and the nuclear membrane fraction of the 25%/45% sucrose density interface were notably deficient in CP49. Vimentin immunoreactive peptides were found in both urea-soluble and urea-insoluble proteins of membrane fractions from the cortex only. Vimentin was particularly enriched in the cortical non-sedimenting membrane fraction. The urea-insoluble filensin immunoreactive peptides were only partially removed by alkali extraction, indicating a very avid association with the membrane. Two dimensional electrophoresis revealed that the urea-soluble protein of the major cortical membrane fraction contained two different filensin-derived 53 kDa fragments. CONCLUSIONS The non-sedimenting membrane fraction, which may reflect a distinct domain of the lens plasma membrane, possesses a membrane-associated cytoskeletal composition different from that of the major sedimenting membrane fractions.

Identification of a Vimentin-Reactive Peptide Associated with Ocular Lens Membranes as Cytokeratin

We wished to identify a 44-kD peptide isolated from lens membrane fractions which has apparent immunoreactivity to antivimentin and to antiphakinin. Urea-soluble proteins from rat lens membrane fractions were separated by two-dimensional electrophoresis, transferred to polyvinylidene fluoride membranes and probed with monoclonal antibodies to vimentin or cytokeratins, or a polyclonal antibody to phakinin. The monoclonal antibody to vimentin recognized the expected protein (Mr = 56 kD, pI = 5.1) and several smaller and more acidic peptides including a 44-kD spot with a pI of 4.9. A similar pattern of immunoreactivity was found with commercially available purified vimentin. The polyclonal antibody to phakinin recognized the expected protein (Mr = 52 kD, pI = 5.0) and several smaller and more acidic peptides, including a 44-kD spot with a pI of 4.9. Matrix-assisted laser desorption ionization mass spectroscopy analysis of the tryptic digest of the spot corresponding to 44 kD and pI of 4.9 identified neither vimentin nor phakinin but did identify peptides derived from ovalbumin (added to samples as an internal standard) and cytokeratin 1. Antibodies which recognize cytokeratin 1 reacted weakly with a 44-kD peptide which comigrated with the 44-kD vimentin-immunoreactive peptide in adjacent lanes of single-dimension SDS polyacrylamide gels. The 44-kD acidic (pI = 4.9) peptide which reacts with a monoclonal antibody to vimentin may be derived from cytokeratin 1.

Specific restriction of cholesterol from cortical lens gap junctional membrane in the U18666A cataract

We have hypothesized that the cholesterol synthesis inhibitor, U18666A, induces nuclear cataracts in the rat by restricting the sterol content of the lens plasma membrane and, therefore, disrupting the structure of gap junctions. In order to directly examine this hypothesis, we isolated total plasma membrane and plasma membrane enriched in gap junctions from the cortical and nuclear regions of lenses from control and U18666A-treated rats. The protein, phospholipid and sterol compositions of the membrane fractions were determined and compared. U18666A treatment resulted in decreased sterol concentrations of both membrane fractions isolated from both the cortical and nuclear regions. The sterol content of total plasma membrane from the cortex and from the nucleus was decreased by 57% and 36% respectively. The sterol content of the gap junctional membrane (membrane domain enriched in gap junctions) from the cortex and from the nucleus was decreased by 71% and 43% respectively. The observation of a selective decrease in the total sterol content of the cortical gap junctional membrane was reinforced by finding a 50% decrease in the sterol/phospholipid molar ratio of this fraction. The corresponding decrease in the sterol/phospholipid ratio of cortical total plasma membrane was only 22%. The sterol/phospholipid ratio of nuclear total plasma membrane was slightly increased (16%), and the sterol/phospholipid ratio of nuclear gap junctional membrane was decreased by only 8%. These data suggest to us that inhibition of cholesterol synthesis in the rat lens by U18666A results in a specific restriction of cholesterol availability for the synthesis of gap junctional membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate-dependent, trifluoperazine-sensitive Ca2+ efflux from rat liver mitochondria. Modulation by a cytosol factor.

Mitochondria possess separate pathways for the uptake and release of Ca2+: an electrophoretic uniporter for Ca2+ influx, and an independent electroneutral efflux mechanism operational when a high membrane potential is retained (reviews [ 1,2]). Ca2+ efflux from heart and brain mitochondria appears to be directly coupled to Na’ entry [3,4] ; in liver mitochondria, however, the mechanism(s) which regulates the release (or retention) of Ca2+ is unclear. The role of Pi in this process is uncertain as, under diverse experimental conditions, Pi has been reported to stimulate [5], inhibit [6] or efflux in parallel with Ca2+ efflux [7]. Hepes and ruthenium red were obtained from Sigma. Ruthenium red was further purified according to [8]. Calmodulin was obtained from Calbiochem-Behring Corp., and trifluoperazine was a gift from Smith Kline and French (Philadelphia PA). Sprague-Dawley rats (175-250 g) were used without fasting. Unless noted otherwise, all procedures were carried out at 4°C. Livers were homogenized as in [9] in 70 mM sucrose, 220 mM mannitol containing 10 mM Hepes (pH 7.4) (SMH). The mitochondrial fraction was isolated by differential centrifugation [9] and washed twice by resuspension in SMH and centrifugation at 95 000 X g. The cytosol was obtained by centrifugation of the post-mitochondrial supernate at 1OOOOOXgfor 1 h. These apparently conflicting reports regarding Pi prompted us to re-evaluate its effect on Ca2+ efflux in liver mitochondria. In our hands, Pi stimulated mitochondrial Ca2+ efflux. We have discovered a heatstable factor in liver cytosol which significantly enhanced Pi-dependent Ca2+ efflux. This enhanced Ca2+ effIux was effectively blocked by the phenothiazine antipsychotic drug, TFP. These results suggest involvement of a cytosol factor in the modulation of Ca” release from rat liver mitochondria.

Lens membrane fraction associated intermediate filaments of different aged rats.

Purpose. To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. Methods. Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. Results. Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. Conclusions. The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane–cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.

Ca2+ release from energetically coupled tumor mitochondria.

A Na+/Ca2+ exchange activity for Ca2+ efflux has been identified in isolated Ehrlich ascites tumor mitochondria. Further, under conditions favoring cycling of Ca2+ across the mitochondrial inner membrane, extramitochondrial [Ca2+] also was shown to be Na+-dependent. The Na+/Ca2+ exchange showed sigmoidal kinetics with a mean (+/-SD) [Na+] required for half maximal stimulation of Ca2+ efflux of 8.4 +/- 3.8 mM and a Hill coefficient of 1.6. Na+/Ca2+ exchange was very sensitive to inhibition by the Ca2+ antagonist diltiazem (56% inhibition at 7.5 nmoles X mg protein-1) whereas a number of other compounds, including verapamil, nupercaine, and trifluoperazine were less effective in inhibiting Ca2+ efflux. These data demonstrate for the first time the presence of a pathway in tumor mitochondria for unidirectional Ca2+ efflux induced by Na+, and provide a mechanism for regulation of tumor intra- and extramitochondrial [Ca2+]. Results of the present study support the need for further study of intracellular Na+ and its role in regulation of Ca2+ homeostasis in tumor cells.

Monoclonal antibody 10A5 recognizes an antigen unique to the water-insoluble 25/45 membrane fraction of the rat ocular lens

The water-insoluble 25/45 fraction and non-sedimenting membrane fraction (NSMF) are two membrane preparations isolated from the ocular lens. The fractions are postulated to represent distinct subdomains of the lens with unique functions. However, attempts to distinguish between the two fractions by detecting proteins present in one fraction but absent from other have been unsuccessful. In this study, we exploited the ability of the mouse immune system to detect antigenic differences between the 25/45 fraction and NSMF isolated from the lenses of 20-day-old rats. We generated a monoclonal antibody (MAb 10A5) that reacts with a ganglioside-like antigen that is present in the 25/45 fraction but absent from the NSMF. Restriction of the antigen to the 25/45 fraction in 20-day-old animals supports the hypothesis that the 25/45 fraction and NSMF represent different subdomains within the ocular lens.