Characterization of membrane steroid binding protein mRNA and protein in lens epithelial cells.

Epithelial cells of the ocular lens contain a 28 kDa membrane protein which is proposed to mediate high affinity binding of steroid hormones and rapid non-genomic actions of steroid hormones. It has been named membrane steroid binding protein (MSBP). Our purpose was to further characterize this protein from cultured bovine lens epithelial cells (BLEC) and compare it to similar forms of the protein present in other species and tissues. The size of the protein's mRNA was examined by Northern blot analysis using a digoxigenin-labelled antisense riboprobe. The sequence of the mRNA was obtained by RT-PCR amplification of poly A+ RNA recovered from cultured BLEC. PCR amplification was conducted using three sets of nested sense and antisense primers, one set at a time. The amino acid sequence of the lens protein was deduced from the revealed cDNA sequence. The hydropathy of the protein was examined by Kyte-Doolittle plots. The sequence of the lens protein's cDNA (about 1.7 kb total) described an open reading frame of 582 residues which coded for a protein of 194 amino acids. The presence of a C-terminal isoprenylation motif suggested by earlier work was not found in the coding region. The deduced amino acid sequence of the lens protein was extremely similar to those of other species and tissues, being 95-98% homologous with that of the other members. All of the MSBPs apparently contain a single membrane spanning domain in the amino terminal. The highly conserved nature of this protein implies a useful function to the cell. We speculate that the protein is a receptor which mediates rapid actions of steroids on lens epithelial cells, such as calcium mobilization, and that the protein plays a role in the mechanism of steroid induced cataracts.

Specific labeling of lens aldehyde dehydrogenase class 1 from (3)H-cholesterol or its derivatives.

We investigated the possibility that sterols could covalently modify ocular lens cell proteins. Incubation of cultured bovine lens epithelial cells (BLEC) with (3)H-cholesterol led to the labeling of a cytosolic protein of about 52 kD. Two-dimensional electrophoresis of the BLEC soluble proteins and fluorography revealed one labeled protein of 52 kD, pI = 6.6, plus a weakly labeled, slightly more acidic protein of the same size. MALDI-MS analysis of both proteins recovered from duplicate gels indicated both to be aldehyde dehydrogenase class 1 (ALDH-1). The identity was confirmed by immunoprecipitation with antiserum to ALDH-1. Alkaline hydrolysis of (3)H-labeled ALDH-1 released most of the radiolabel as compounds much more polar than cholesterol. We speculate that lens ALDH-1 can participate in the oxidation of cholesterol or its derivatives to unidentified sterol carboxylic acids and that the labeled protein reflects capture of ALDH-1 with sterol intermediates covalently bound to the enzyme in ester linkage. Lens ALDH-1 might, therefore, participate in the detoxication of polar sterols.

Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses.

Previous studies in our laboratory have provided direct evidence for the existence of distinct cholesterol domains within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P) mole ratios ranging from 1 to 4. Since membrane cholesterol content is disturbed in the development of cataracts, it was hypothesized that perturbation of cholesterol domain structure occurs in cataracts. In this study, fiber cell plasma membranes were isolated from both normal (control) and cataractous lenses and assayed for cholesterol and phospholipid. Control and cataractous whole lens membranes had C/P mole ratios of 3.1 and 1.7, respectively. Small angle x-ray diffraction approaches were used to directly examine the structural organization of the cataractous lens plasma membrane versus control. Both normal and cataractous oriented membranes yielded meridional diffraction peaks corresponding to a unit cell periodicity of 34.0 A, consistent with the presence of immiscible cholesterol domains. However, comparison of diffraction patterns indicated that cataractous lens membranes contained more pronounced and better defined cholesterol domains than controls, over a broad range of temperature (5-40 degrees C) and relative humidity (52-92%) levels. In addition, diffraction analyses of the sterol-poor regions of cataractous membranes indicated increased membrane rigidity as compared with control membranes. Modification of the membrane lipid environment, such as by oxidative insult, is believed to be one potential mechanism for the formation of highly resolved cholesterol domains despite significantly reduced cholesterol content. The results of this x-ray diffraction study provide evidence for fundamental changes in the lens fiber cell plasma membrane structure in cataracts, including the presence of more prominent and highly ordered, immiscible cholesterol domains.

Retinal structure and function in an animal model that replicates the biochemical hallmarks of desmosterolosis.

Desmosterolosis is a rare, autosomal recessive, human disease characterized by multiple congenital anomalies in conjunction with grossly elevated levels of desmosterol and markedly reduced levels of cholesterol in all bodily tissues. Herein, we evaluated retinal sterol composition, histology, and electrophysiological function in an animal model that exhibited the biochemical features of desmosterolosis, produced by treating pregnant rats and their progeny with U18666A, an inhibitor of desmosterol reductase. Treated rats had cataracts, were substantially smaller, and had markedly high levels of desmosterol and profoundly low levels of cholesterol in their retinas and other tissues compared to age-matched controls. However, their retinas were histologically normal and electrophysiologically functional. These results suggest that desmosterol may be able to replace cholesterol in the retina, both structurally and functionally. These findings are discussed in the context of "sterol synergism".

Cholesterol synthesis in the vertebrate retina: effects of U18666A on rat retinal structure, photoreceptor membrane assembly, and sterol metabolism and composition.

Treatment of neonatal rats with U18666A, an inhibitor of desmosterol delta24-reductase, results in accumulation of desmosterol (delta5,24) and depletion of cholesterol (delta5) in various bodily tissues and also causes cataracts. We evaluated the effects of U18666A on the sterol composition, de novo sterol synthesis, and histological structure of the retina. Neonatal Sprague-Dawley rats were injected subcutaneously with U18666A (15 mg/kg, in olive oil ) every other day from birth through 3 wk of age; in parallel, control rats received olive oil alone. At 21 d, treated and control groups each were subdivided into two groups: one group of each was injected intravitreally with [3H]acetate; retinas were removed 20 h later and nonsaponifiable lipids (NSL) were analyzed by radio-high-performance liquid chromatography. The other group was injected intravitreally with [3H]leucine; 4 d later, one eye of each animal was evaluated by light and electron microscopy and light microscopic autoradiography, while contralateral retinas and rod outer segment (ROS) membranes prepared therefrom were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. In the treated group, the delta5/delta5,24 mole ratio of retinas was ca. 1.0, and >88% of the NSL radioactivity was in delta5,24; in contrast, control retinas had delta5/delta5,24 >170, with >80% of the NSL radioactivity in delta5. Retinal histology, ultrastructure, ROS renewal rates, and rhodopsin synthesis and intracellular trafficking were comparable in both treated and control animals. These results suggest that desmosterol can either substitute functionally for cholesterol in the retina or it can complement subthreshold levels of cholesterol by sterol synergism.

Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes.

The molecular structure of human ocular lens fiber cell plasma membranes was examined directly using small angle x-ray diffraction approaches. A distinct biochemical feature of these membranes is their high relative levels of free cholesterol; the mole ratio of cholesterol to phospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization of cholesterol in this membrane system is not well understood, however. In this study, the structure of plasma membrane samples isolated from nuclear (3.3 C/P) and cortical (2.4 C/P) regions of human lenses was evaluated with x-ray diffraction approaches. Meridional diffraction patterns obtained from the oriented membrane samples demonstrated the presence of an immiscible cholesterol domain with a unit cell periodicity of 34.0 A, consistent with a cholesterol monohydrate bilayer. The dimensions of the sterol-rich domains remained constant over a broad range of temperatures (5-20 degrees C) and relative humidity levels (31-97%). In contrast, dimensions of the surrounding sterol-poor phase were significantly affected by experimental conditions. Similar structural features were observed in membranes reconstituted from fiber cell plasma membrane lipid extracts. The results of this study indicate that the lens fiber cell plasma membrane is a complex structure consisting of separate sterol-rich and -poor domains. Maintenance of these separate domains may be required for the normal function of lens fiber cell plasma membrane and may interfere with the cataractogenic aggregation of soluble lens proteins at the membrane surface.

Lens epithelia contain a high-affinity, membrane steroid hormone-binding protein.

PURPOSE: To describe the serendipitous discovery of a high-affinity, membrane steroid-binding protein (MSBP) in lens epithelial cells and to examine the binding of progesterone to epithelial cell membranes. METHODS: Bovine lens epithelial cells (BLECs) were cultured in media containing 3H-mevalonolactone to examine protein prenylation by mevalonate-derived isoprenes. Cell proteins were divided into insoluble and soluble fractions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and label detected by fluorography. Insoluble proteins were then fractionated on a C18 reversed-phase column. A high-performance liquid chromatography fraction containing a 28kDa 3H-labeled hydrophobic protein was collected, lyophilized, and subjected to SDS-PAGE and the separated proteins transferred to membrane. Protein in the recovered 28-kDa band was submitted for identification by N-terminal sequence analysis. Microsomal membranes prepared from fresh epithelia of intact bovine, rat, and human lens and cultured BLECs were tested for the presence of MSBP by western blot analysis using an antiserum to porcine liver microsomal MSBP. Radiolabeling of MSBP from 3H-mevalonate was confirmed by immunoprecipitation using the same antiserum. 3H-Progesterone was incubated with microsomal membrane from bovine lens epithelia to measure high-affinity binding. Radiolabeled progesterone-protein complexes were trapped on glass filters and radioactivity measured and the binding data subjected to Scatchard analysis. RESULTS: Membrane recovered from BLECs incubated with 3H-mevalonolactone contained a 3H-labeled 28-kDa protein fraction. The N-terminal sequence of the principal protein in this fraction was very similar to that of the recently discovered MSBP. Western blot analysis with antiserum to MSBP indicated the presence of the 28-kDa protein in the microsomal fraction from BLECs and epithelia of bovine, rat, and young human lenses but not in lens fiber cell membrane. Microsomal membrane from intact bovine lens epithelium bound progesterone with high affinity, with disso ciation constant (Kd) at approximately 75 nM and a receptor concentration of approximately 3 picomoles/mg protein. CONCLUSIONS: The lens epithelium contains a 28-kDa membrane protein that can bind progesterone and perhaps other steroid hormones with high affinity. The protein appears to be microsomal and prenylated. The MBSP may mediate rapid nongenomic steroid effects that contribute to steroid-induced cataracts.

Human lens cholesterol concentrations in patients who used lovastatin or simvastatin.

OBJECTIVE: To determine whether long-term therapeutic use of the hypocholesterolemic drugs lovastatin and simvastatin significantly alters the distribution and concentration of cholesterol in the human lens. Such changes might precede observable alterations in lens structure. METHODS: Pairs of lenses (9-13 pairs) from patients (age range, 46-81 years) who had been taking lovastatin or simvastatin before their death (estimated for the previous 2-4 years) and lenses from similarly aged control subjects were divided into outer cortex and inner cortex plus nucleus by dissolution in a detergent-containing buffer. Ten minutes of dissolution removed 17% to 19% of the lens total volume, which accounted for about 20% of the width of the equatorial cortex and 75% of the width of the sagittal cortex. This fraction plus the residual lens was homogenized, saponified, and assayed for cholesterol by gas-liquid chromatography. RESULTS: The cortex of adult control lenses contained about 4 microg of cholesterol per cubic millimeter of volume. This concentration increased to 10 to 15 microg/mm3 in the adult nucleus and decreased to about 6 microg/mm3 in the juvenile and fetal nucleus. Treatment with neither lovastatin nor simvastatin significantly altered the concentration of cholesterol in either the cortex or nuclear fractions. CONCLUSIONS: Variations in concentration of cholesterol along the radii of the lens reflect differences in the density or packing of fiber cell membranes. The observed distribution of cholesterol supports the recent model of the adult lens structure, which, from surface to center, is the cortex, adult nucleus,juvenile nucleus, fetal nucleus, and embryonic nucleus. Finding no significant changes in concentration of cholesterol in the cortex formed during treatment with lovastatin or simvastatin reinforces the results of clinical studies that indicate a high lenticular safety of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Nevertheless, caution is encouraged in assuming a similar ocular safety in newer drugs that inhibit cholesterol synthesis at later metabolic steps. CLINICAL RELEVANCE: Does clinical use of hypocholesterolemic drugs alter lens cholesterol?

Intense myristoylation of a single protein in the ocular lens.

A single protein of the ocular lens was intensely myristoylated following short term incubation of cultured bovine lens epithelial cells and intact rat lenses with 3H-myristic acid. It was acidic (pI <5), about 19 kDa and present exclusively in the cytosol of both cultured epithelial cells and the epithelium of the young rat lens. Fiber cell proteins were not labeled. The myristoylated protein was not seen in the epithelium of the adult rat. Essentially no protein mass was evident in the 19-20 kDa range when samples of the labeled-soluble protein were fractionated by either HPLC coupled with SDS-PAGE or 2D-electrophoresis. These findings suggest that the myristoylated-soluble protein of 19 kDa in lens (p19L) is a rapidly-turning over minor protein likely associated with lens growth. The absence of any apparent membrane association for a myristoylated protein appears unusual. The trace nature of p19L has frustrated attempts at its identification by MALDI-MS.

Prenylation of proteins by the intact lens.

PURPOSE: To describe and identify proteins prenylated by the intact rat lens. METHODS: Lenses from young rats were incubated for 24 hours in TC199 medium containing 22 microM lovastatin and 110 microCi/ml [3H]mevalonolactone. Proteins of the epithelium and fiber cells were separated by high-performance liquid chromatography (HPLC) and one- and two-dimensional electrophoresis, and the 3H label was detected by fluorography. Treatment of labeled proteins with methyl iodide released isoprenes that were identified by HPLC analysis. The identity of the prenylated proteins was probed by N-terminal sequence analysis and matrix-assisted, laser desorption ionization-mass spectrometry (MALDI-MS). RESULTS: The pattern of protein prenylation by epithelial and fiber cells was similar. Most of the labeled proteins were water insoluble and were assumed to be membrane associated. A group of 21-kDa to 29-kDa proteins were most intensely labeled and were modified mainly with geranylgeranyl. A highly labeled, approximately 80-kDa insoluble protein and a lesser labeled, 64-kDa soluble protein were the only other significant prenylated proteins. Both were farnesylated. MALDI-MS analysis suggested that the 80-kDa protein is a cytokeratin. N-terminal sequence analysis indicated that the 64-kDa soluble protein is beta-tubulin. CONCLUSIONS: A limited set of proteins are prenylated by the young rat lens. The 21-kDa to 29-kDa proteins were modified mainly by geranylgeranyl and are likely members of the numerous small GTP binding proteins. The authors express caution about accepting the identities of the 64-kDa and 80-kDa proteins as beta-tubulin and cytokeratin-1, respectively--proteins in these families do not contain the required CAAX motif. The 80-kDa farnesylated protein could be novel and unique to the lens, because no farnesylated protein of this size has been previously reported.

Influence of cholesterol on the interaction of alpha-crystallin with phospholipids.

The influence of cholesterol on the binding of alpha-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of alpha-crystallin to membranes. In the absence of cholesterol, binding of alpha-crystallin liposomes composed of either sphingomyelin, disteroyl-phosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE. Since this fluorescence probe resides in the polar headgroup region of the membrane, the observed changes indicated that the binding of alpha-crystallin affected the structure of these membrane regions. The ability of alpha-crystallin to modulate membrane structure suggests yet another potential role for this lens protein. Addition of cholesterol markedly decreased the binding of alpha-crystallin to liposomes composed of either sphingomyelin or disteroylphosphatidylcholine and antagonized the capacity of bound alpha-crystallin to decrease membrane surface order. This antagonism could be explained by the ability of cholesterol to directly decrease the anisotropy of the fluorophore in sphingomyelin membranes unexposed to alpha-crystallin. Thus, with cholesterol present, a further decrease in membrane order upon subsequent binding of alpha-crystallin was less likely. The results obtained with the sphingomyelin liposomes are considered most meaningful, since sphingomyelins are the principal phospholipids in the human lens nuclear membrane and cholesterol preferentially interacts with sphingomyelin. We conclude that cholesterol in lipid membranes can antagonize the binding of alpha-crystallin and thus interfere with the capacity of bound alpha-crystallin to alter membrane order. We suggest that such actions of cholesterol might serve to preserve lens membrane structure in the physiological state where the concentration of soluble alpha-crystallin is great.

Probing cataractogenesis associated with mevalonic aciduria.

PURPOSE: Mevalonic aciduria in humans results from a genetic deficiency of mevalonate kinase and is characterized by very high plasma mevalonic acid levels, developmental malformations and cataracts. This study tested the possibility that the cataracts could result from direct toxicity of the accumulated mevalonate. METHODS: Young rat lenses were cultured for up to 4 days in medium TC199 containing 1 to 5 mM mevalonic acid. Changes in the water, sodium and potassium content of the lens were followed; electrolytes were measured by atomic absorption spectroscopy. The identities of proteins leaked from the lens were determined by sodium dodecylsulfate polyacrylamide electrophoresis and isoelectric focusing. Changes in cation flux were measured by 86Rb uptake. Lens concentrations of mevalonic acid were measured from uptake of 3H-mevalonolactone. RESULTS: Culture of young rat lenses with 3 to 5 mM mevalonic acid produced lens opacification and nuclear cataracts starting within 1 to 2 days of culture. Mevalonic acid did not concentrate in the lens. Treated lenses accumulated water and sodium and lost potassium and soluble gamma crystallin proteins. These changes were preceded by a loss of the len's capacity to concentrate 86Rb, a potassium analogue. The loss of 86Rb uptake might have been due to a slow poisoning of the cation pump, direct effects on membrane integrity or both. CONCLUSIONS: The results show that chronic exposure of the lens to mevalonic acid can induce cataracts, which appear caused by a progressive increase in the permeability of lens cell membranes. The cataracts associated with mevalonic aciduria could be due to toxicity from mevalonic acid.

Posttranscriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in lens epithelial cells by mevalonate-derived nonsterols.

The ocular lens must continuously synthesize the cholesterol required to support membrane formation for its life-long growth. The roles of transcriptional and posttranscriptional mechanisms in controlling 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) protein levels in cultured lens epithelial cells were examined by measuring the effect of restricting exogenous cholesterol, endogenous cholesterol synthesis and mevalonate derived nonsterols upon HMGR protein and mRNA levels and upon the synthesis and degradation of HMGR protein. Sterols were restricted by culturing in lipoprotein deficient media and blocking 2,3-oxidosqualene cyclase with U18666A. Mevalonate derived nonsterols were additionally restricted by inhibition of HMGR activity with lovastatin. A 4-fold increase in HMGR protein levels due to restricting sterols with U18666A could be explained by comparably increased mRNA levels and enzyme protein synthesis. The very rapid turnover of HMGR protein (T(1/2) approximately 45 min) was unaffected. The additional restriction of mevalonate derived nonsterols increased HMGR protein levels to about 400-fold. A 10-fold slowing in the rate of enzyme degradation coupled with at least a 5-fold increase in mRNA levels likely accounted for this accumulated protein mass. The capacity of the nonsterol regulators to promote enzyme degradation appeared independent of sterols, since mevalonate restored rapid degradation of HMGR protein when 2,3-oxidosqualene cyclase activity was simultaneously blocked. Thus, in cultured lens epithelial cells, sterols appear to exert a modest influence on HMGR protein levels solely by suppressing transcription; whereas, mevalonate derived nonsterols exert major influence mainly by accelerating enzyme protein degradation. We speculate that nonsterol isoprenes might be important for preventing overexpression of cholesterol biosynthesis in the intact lens.

Properties of alpha-crystallin bound to lens membrane: probing organization at the membrane surface.

Alpha crystallin, one of the three major soluble proteins of the eye lens, appears to be a natural extrinsic protein of lens plasma membrane. Membrane-immobilized alpha-crystallin could provide a template for the increased association of protein with lens membrane seen in aging and cataracts. Alpha-crystallin binds to lens membrane through both a high-affinity saturable and low-affinity nonsaturable process. The organization of alpha-crystallin at the membrane surface was proved by the examination of various chemical reactivities and a functional property of the membrane bound protein. The carboxyl-terminal domain of membrane bound alpha-crystallin appeared to be as readily cleaved by mild trypsinolysis as that of the soluble protein and the cleaved protein remained bound to the membrane. The immobilized protein was more extensively crosslinked by a bifunctional primary amine-reactive agent than the soluble protein. No evidence for crosslinking to membrane intrinsic protein was obtained. Like soluble alpha-crystallin, the membrane bound protein displayed chaperone-like activity, a property dependent upon quaternary structure. These findings were interpreted to indicate that alpha-crystallin binds to lens membrane as an aggregate with only a fraction of each aggregate in direct contact with the membrane's hydrophobic surface. It is suggested that the nonsaturable binding reflects low affinity association of soluble alpha-crystallin with a layer of membrane-immobilised protein.

Neutral lipids of the plasma membrane: composition of plasma membrane fractions isolated from ocular lens.

PURPOSE: To quantitatively examine and compare the neutral lipid composition of sedimenting and non-sedimenting ocular lens plasma membrane fractions isolated from the cortex and nucleus. METHODS: Homogenates of bovine lens cortical and nuclear regions were subjected to centrifugation to sediment the water insoluble fractions, which were used as the sedimenting membrane fractions. The non-sedimenting membrane fractions were isolated from the water soluble fractions by flotation during overnight, high density centrifugation. Lipids were extracted from the membrane fractions and neutral lipids were separated by thin layer chromatography. RESULTS: A greater concentration of triacylglycerol was found in the whole lens non-sedimenting membrane fraction (10.6 micrograms triacylglycerol/mg phospholipid) than in the sedimenting membrane (3.7 micrograms triacylglycerol/mg phospholipid). Triacylglycerol was found in the non-sedimenting membrane fraction isolated from both lens cortex and nucleus, but was not detected in the water insoluble fraction isolated from the lens nucleus. The higher triacylglycerol concentration of the non-sedimenting membrane correlates with its lower cholesterol level. Free fatty acids were found in both the whole lens non-sedimenting membrane fraction (66.2 micrograms free fatty acid/mg phospholipid) and water insoluble fraction (40.3 micrograms free fatty acid/mg phospholipid). Free fatty acids were more abundant in the nucleus. This could reflect phospholipase attack of nuclear phospholipids, a mechanism proposed to explain the lower glycerophospholipid concentration in this region. The fatty acid compositions of the phospholipids from the non-sedimenting and sedimenting membrane fractions were essentially identical. Triacylglycerols (from both non-sedimenting and sedimenting membrane fractions) contained a greater proportion of saturated fatty acids and a lesser proportion of mono-unsaturated fatty acids than the phospholipids. The triacylglycerols of the non-sedimenting membrane fraction contained a greater amount of palmitic and lesser amounts of myristic and stearic acids than the triacylglycerols of the sedimenting membrane fractions. The free fatty acids (of both non-sedimenting and sedimenting membrane fractions) were composed of a greater proportion of saturated fatty acids and a lesser proportion of mono-unsaturated fatty acids than found in the phospholipids. The non-sedimenting membrane fraction of both cortex and nucleus was enriched with an unidentified (presumed) lipid. These findings support earlier work indicating most membrane triacylglycerols are enriched in saturated fatty acids. CONCLUSIONS: These results demonstrate differences in the neutral lipid composition between non-sedimenting and sedimenting lens membrane fractions and furthers our hypothesis that the non-sedimenting membrane could represent a specialized domain of the lens plasma membrane.

Role of cholesterol in the structural order of lens membrane lipids.

Cholesterol may order or disorder phospholipids. The physiological contribution of cholesterol to the structural order of lens membrane lipids was determined. Cholesterol and phospholipid from bovine lens nuclear and cortical tissue were separated by thin layer chromatography. The effect of cholesterol upon the trans to gauche transition of the hydrocarbon chains was assessed by measuring CH2 infrared stretching band frequencies as cholesterol was added back to the phospholipids. Although the relative cholesterol level of nuclear lipid was much higher than that of the cortex (59 vs. 36 mol%, respectively), the structural order of unfractionated nuclear and cortical lipids were similar at physiological temperature. Cholesterol added to lipids devoid of cholesterol produced a sharp biphasic effect on the structural order of nuclear lipids, increasing the trans conformation from 56% to 0 mol% cholesterol to 74% at 18% cholesterol to 41% trans at 59 mol% cholesterol. Cholesterol addition produced a shallow biphasic change in the percentage trans conformation of cortical lipids. Maximum order (about 40% trans conformation) was seen at a cholesterol level equal to that of intact cortical lipid (36 mol%). The physiological role of cholesterol is to increase the structural order of cortical membrane lipid and decrease order in nuclear lipid. The net result is a similarity in the structural order of cortical and nuclear membrane. We suggest that the different response of cortical and nuclear lipids to added cholesterol is linked to differences in the phospholipid composition between these two lens regions. In the absence of cholesterol, nuclear phospholipids are much more highly ordered than those of the cortex.

Cholesterol and cataracts.

Inherited defects in enzymes of cholesterol metabolism and use of drugs which inhibit lens cholesterol biosynthesis can be associated with cataracts in animals and man. The basis of this relationship apparently lies in the need of the lens to satisfy its sustained requirement for cholesterol by on-site synthesis, and impairing this synthesis can lead to alteration of lens membrane structure. Lens membrane contains the highest cholesterol content of any known membrane. The Smith-Lemli-Opitz syndrome, mevalonic aciduria, and cerebrotendinous xanthomatosis all involve mutations in enzymes of cholesterol metabolism, and affected patients can develop cataracts. Two established models of rodent cataracts are based on treatment with inhibitors of cholesterol biosynthesis. The long-term ocular safety of the very widely used vastatin class of hypocholesterolemic drugs is controversial. Some vastatins are potent inhibitors of cholesterol biosynthesis by animal lenses, can block cholesterol accumulation by these lenses and can produce cataracts in dogs. Whether these drugs inhibit cholesterol biosynthesis in human lenses at therapeutic doses is unknown. Results of clinical trials of 1-5 years duration in older patient populations indicate high ocular safety. However, considering the slow life-long growth of the lens and its continuing need for cholesterol, longterm safety of the vastatins should perhaps be viewed in units of 10 or 20 years, particularly with younger patients.

Role of transcription, translation, and protein turnover in controlling the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the lens.

PURPOSE: To determine the principle site (epithelium or superficial cortex) of gene transcription and mRNA translation for the regulatory enzyme of lens cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). To evaluate the contribution of waning enzyme synthesis versus enzyme turnover by proteolysis in accounting for the disappearance of HMGR protein from elongated fiber cells. METHODS: Young rats were treated with lovastatin, a drug that increases transcripts of the HMGR gene and translation of HMGR mRNA in lens secondary to inhibiting cholesterol biosynthesis. The relative concentration of HMGR mRNA in lens epithelium and superficial cortex was estimated by a competitive reverse transcriptase-polymerase chain reaction system. Relative HMGR protein levels were estimated by Western blot analysis. Because lovastatin is cleared rapidly from the lens, the half-life of HMGR protein in epithelium and cortex was estimated by following the disappearance of the increased pool of enzyme protein from each compartment with time after halting drug treatment. RESULTS: Between 75% and 90% of the total content of HMGR mRNA and protein in the epithelium and the superficial cortex of control rat lens was located in the cortex. Treatment with lovastatin increased the content of the mRNA in epithelium and cortex by approximately 0.4-fold and HMGR protein content approximately 5-fold. Although the concentration of HMGR mRNA and protein was similarly increased in epithelium and superficial cortex, approximately 85% to 90% of the total increase in mRNA and protein content was located in the cortex because of that area's greater mass. The half-life for the disappearance of the increased pool of HMGR protein from epithelium and cortex was similar at approximately 14 to 17 hours. CONCLUSIONS: The bulk of HMGR gene transcription and mRNA translation apparently is confined to elongating fiber cells. The 10-fold greater increase in enzyme protein than mRNA levels after lovastatin treatment indicates that enzyme concentration in lens is controlled mainly by effects on HMGR mRNA translation or rates of HMGR proteolysis. The observed rapid turnover of enzyme protein in the epithelium and the superficial cortex, if applicable to the deeper cortex and the homeostatic state (absence of drug exposure), suggests that the gradual disappearance of HMGR protein from the lens could be caused by waning of enzyme synthesis rather than to proteolysis in the absence of continuing enzyme synthesis.

Protein associated with human lens 'native' membrane during aging and cataract formation.

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.