Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Highly selective membranes in protein ultrafiltration

A new ultrafiltration technique based on a multimembrane stack has been developed to fractionate solutes closer in size than conventionally possible. The technique is illustrated here by obtaining a pure protein product from a binary protein mixture. By employing membranes in series without any gaskets or spacers in-between, ultrafiltration is carried out to separate two proteins relatively close in molecular weight or size. Flat YM30 regenerated cellulose membranes, all of the same molecular weight cut-off (MWCO) 30,000, are stacked together in the desired number, and ultrafiltration takes place. The membrane rejection of a protein is amplified with each additional membrane, ultimately resulting in a completely rejected species. Complete purification of the more permeable protein may be achieved regardless of the physicochemical condition that may be optimal or suboptimal for selective separation by a single membrane. Two systems, myoglobin and beta-lactoglobulin, as well as myoglobin and alpha-lactalbumin were studied, under various operating conditions. The solvent flux reduction encountered when each membrane is added may also be avoided, by operating at increased pressure, while still achieving the desired purification. Cleaning in situ is achievable with reproducible experimental results before and after on-line cleaning. The results clearly demonstrate that multimembrane stacks can be used for fractionation of proteins that are quite close in molecular weight/size.     

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.     

Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes

Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.     

VIP receptors from porcine liver: high yield solubilization in a GTP-insensitive form.

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-lactam antibiotics from rabbit small intestinal brush border membranes

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-lactam antibiotics with an apparent molecular weight of 127,000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the alpha-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and beta-lactam antibiotics of apparent molecular weight 127,000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active beta-lactam antibiotics across the enterocyte brush border membrane.     

The major soluble cytochromes of the obligately aerobic sulfur bacterium,Thiobacillus neapolitanus

Four cytochromes were isolated from soluble extracts of the aerobic sulfur bacterium, Thiobacillus neapolitanus. The two most abundant proteins were purified to homogeneity and thoroughly characterized. Cytochrome c-554 (547) is a monomeric, small molecular weight protein which is unusual in having two well-resolved alpha peaks in UV-visible absorption spectra. The redox potential is 208 mV. Native cytochrome c-549 is oligometric, but has a subunit size of about 26.000. The yield of this protein could be improved dramatically by washing membranes with 30% ammonium sulfate, but the material solubilized by this method had a larger native molecular weight than that in the initial 0.1 M Tris-Cl extract and behaved differently on chromatography. The properties of cytochrome c-549 including subunit size and UV-visible absorption spectra are similar to mitochondrial cytochrome c ₁ and chloroplast cytochrome f, which suggests that it may be a modified form of the predominant membrane cytochrome. Based on cytochrome content, it is suggested that T. neapolitanus is not closely related to other thiobacilli.Dedicated to Prof. Dr. G. Drews on the occasion of his sixtieth birthday     

Phosphorylation of thylakoid proteins by a purified kinase

A simplified method is given for the purification of a 64-kilodalton protein kinase from spinach or pea thylakoid membranes (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385). In a heterogeneous reconstitution system comprised of purified kinase and washed thylakoids (having their intrinsic kinase inactivated or removed), endogenous light-harvesting pigment protein of photosystem II could serve as a substrate. Its phosphorylation did not require rebinding of kinase to the thylakoid membrane and, like the phosphorylation of solubilized pigment protein, was not under redox control. No reconstitution was observed upon replacing 64-kilodalton protein kinase with 25-kilodalton protein kinase (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 14062-14068). Tryptic digestion of phosphorylated membranes removed the site of phosphorylation; the phosphorylated amino acid present in light-harvesting pigment protein and its tryptic peptide was threonine. Immunoglobulin from a polyclonal antiserum, raised against the purified enzyme, fully inhibited kinase activity toward solubilized and endogenous pigment protein. At higher titers, the antibody was effective in totally inhibiting the redox-sensitive phosphorylation of thylakoid proteins by endogenous kinase; inhibition profiles for phosphorylation of pigment protein and thylakoid proteins of 32, 16, and 9 kilodaltons were essentially identical. The 64-kilodalton protein kinase would thus appear to be responsible for all of the observed phosphorylation of thylakoid phosphoproteins.     

Membrane proteins of Escherichia coli K-12: two-dimensional polyacrylamide gel electrophoresis of inner and outer membranes.

Protein compositions of the inner and outer membranes of Escherichia coli K-12 have been analyzed by two-dimensional gel electrophoresis in which proteins are separated according to apparent isoelectric point (first dimension) and to apparent molecular weight (second dimension). Membrane proteins except for a pair of major outer membrane proteins (proteins Ia and Ib) were found to be solubilized effectively by lysis buffer containing urea, Triton X-100, ampholines and 2-mercaptoethanol. The latter two proteins could be solubilized after precipitation of membrane fraction with trichloroacetic acid; they formed a pair of spots at an acidic region on the electropherogram. Another major protein of the outer membrane, protein II, was also identified. Most of the inner and outer membrane proteins were shown to be focused at a pH range between 4 and 6.5. Specific protein patterns characteristic for both the inner and outer membranes could thous be visualized by the present system. At least 120 and 50 protein species were detected for the inner and outer membranes, respectively.     

Purification and characteristics of photoreceptor light-activated guanosinetriphosphatase

We describe a reconstitution of light-activated vertebrate photoreceptor GTPase and a purification of the GTP-binding protein (G protein), which is a component of the GTPase and modulates the light-activated phosphodiesterase (PDE) enzyme system. Rod outer segments (ROS) of bull frogs were treated with ethylenediaminetetraacetic acid (EDTA), and the GTPase and PDE fractions were solubilized (EDTA supernatant). When the EDTA supernatant and EDTA-treated membrane fraction (EDTA-washed membranes) were recombined, light-dependent GTPase activity appeared. In the reconstituted system, the Km for GTP as substrate was 0.5 microM; the optimum pH was 7.5-8.0. The isoelectric point of GTPase in EDTA supernatant was 4.8. G protein was purified 400-fold from ROS, and the molecular weight of G protein was determined to be 40 000 by polyacrylamide gel electrophoresis. The amount of G protein in ROS was calculated as at least 1 molecule per 400 rhodopsin molecules. By recombining (in the presence or absence of GTP) purified G protein, PDE, H fraction (an additional component of GTPase), and illuminated or unilluminated EDTA-washed membranes (as a source of rhodopsin), we showed that illuminated rhodopsin, G protein, PDE, and GTP are the minimum requirements for light-dependent PDE activity. We discuss the significance of these findings in the regulation of the light-activated GTPase and PDE activities, especially with regard to the mechanism of activation.     

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis.

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of protein solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Partial purification and characterization of the low density lipoprotein receptor from bovine adrenal cortex

The low density lipoprotein (LDL) receptor has been solubilized from bovine adrenocortical membranes with octyl-beta-D-glucoside and purified 350-fold in the presence of the detergent. The activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of egg phosphatidylcholine liposomes. the receptor-phosphatidylcholine liposomes bound 125I-LDL with the same affinity and specificity as did the native LDL receptor of intact membranes. The complex of receptor and octylglucoside had a Stokes radius of 53.5 A as determined by agarose gel filtration. The sedimentation coefficient, s20,w, of the receptor . octylglucoside complex was 7.3 as determined by metrizamide density gradient centrifugation. An identical value for the sedimentation coefficient was obtained when deuterium oxide was substituted for water in the metrizamide gradient. These data were used to derive an estimate of 163,000 for the molecular weight of the LDL receptor . octylglucoside complex (range of molecular weight, 152,000 to 170,000). The receptor is an acidic protein as determined by its behavior on ion exchange chromatography. In the most highly purified LDL receptor preparation, which had been subjected to the sequential steps of solubilization, DEAE-cellulose chromatography, agarose gel filtration, and phosphatidylcholine/acetone precipitation, the receptor was estimated to constitute about 5% of the total protein. Thus, complete purification of the LDL receptor from bovine adrenocortical membranes will require an additional 20-fold purification, or a total purification of about 7,000-fold.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.     

Characterization of polyclonal antibodies to cell-surface antigens from ovine adipose tissue.

High titres of polyclonal antibodies to specific proteins of ovine adipose tissue plasma membranes were raised in horses and chickens following repeated injections of purified plasma membranes. Horse antiserum was highly species specific, reacting only weakly with rat adipose tissue plasma membranes. A protein of molecular weight 68,000 was most antigenic in that it was readily precipitated; however proteins of 25,000, 82,000 and 94,000 were also precipitated when the reaction was performed for longer with a higher antiserum concentration. Chicken egg yolk IgY reacted strongly with ovine adipose tissue plasma membranes as did those preparations from horse, but IgY was ineffective in immunoprecipitating solubilized membrane proteins and exhibited no cytotoxic reaction when incubated with intact ovine adipocytes. However, horse antiserum produced a strong complement-dependent cytotoxic reaction with ovine adipocytes, as measured by leakage of lactate dehydrogenase. This work suggests that the membrane protein of molecular weight 68,000 is likely to be an important antigenic marker for ovine adipocytes.     

Eye lens ageing in the dogfish (Mustelus canis).

1. Age-related alterations in the distribution of water-soluble, high molecular weight (colloidal), and water-insoluble proteins of the lens of smooth dogfish (Mustelus canis) were measured. 2. The ages of these animals ranged approx from 2 to 50 yr, during which time the lenses grew from 100 to 1500 mg (wet wt). The lenses contained approx 50% water. 3. Water-insoluble protein accumulated to a level greater than 50% of the total proteins by the time the animals reached maturity. The lenses of other animals, such as mammals and humans, would be opaque if they had a similar insoluble protein content. 4. Each protein fraction contained the same protein chains (mol. et 1900-25,000 daltons), as observed by SDS polyacrylamide gel electrophoresis, except the water-insoluble fraction, which seemed to contain several extra protein chains with higher molecular weights, which represent fiber cell membrane components. 5. Further purification of these fiber cell membranes indicated that their protein chain makeup was mainly from the same low molecular weight chains present in the soluble and high molecular weight colloidal proteins.