Biochemical evaluation of the corneal endothelium

Transcription

1 Isolation of the plasma membrane from corneal endothelial cells Z. Suzanne Zam, James Cerda,* and Frank M. Polack The plasma membranes of normal rabbit endothelail cells were isolated by the use of an aqueous two-phase polymer system. The membrane fraction was identified by electron microscopy, and a fivefold to eightfold increase in the specific activity of two plasma membrane markers, Na + -K + -ATPase and 5'nucleotidase was found. Recovery of the enzyme markers averaged 45% and 22%, respectively. Analysis of the purified membranes for glucose-6- phosphatase, a marker for endoplasmic reticulum, showed no contamination by this structure. This method for cell membrane characterization is promising in determining the enzymatic alterations of diseased corneas. Key words: cornea, corneal endothelium, corneal biochemistry, membrane isolation Biochemical evaluation of the corneal endothelium has primarily been limited to histochemical studies which indicate what reactions are possible but give little or no quantitative information on functional activities. For the most part, such studies have been concerned with the determination of respiratory enzymes. 1 Their presence or absence has been used to determine cell regeneration and injury and to evaluate corneal viability for purposes of transplantation. 2-3 Recently, attempts have been made to study the metabolism of isolated endothelia from rabbit 4 ' 5 and cow 6 eyes in relation to the action of the sodium pump, but our knowledge of the chemical composition and enzymatic activity of this structure remains deficient. From the Departments of Ophthalmology and *Gastroenterology, University of Florida College of Medicine, Gainesville. Supported by grant EY-446 from the National Eye Institute, Bethesda, Md. Submitted for publication Feb. 23, Reprint requests: Frank M. Polack, M.D., Department of Ophthalmology, College of Medicine, Box J-284, JHMHC, Gainesville, Fla Since the cell membrane participates in many cell functions, it seemed important to determine the various enzymatic and structural components in the normal and the diseased cornea. This is of particular interest in endothelial dystrophies where the first signs of cell dysfunction involve corneal edema and may be due to changes in enzymatic activity of altered endothelial cells. The purpose of this paper is to describe the methodology used for the isolation and biochemical analysis of the plasma membrane of rabbit endothelial cells. Materials and methods Membrane isolation. Corneas were obtained from the eyes of adult New Zealand albino rabbits immediately after sacrifice by an intravenous injection of Nembutal. The endothelial cells from four rabbit corneas were removed by scraping with a rubber policeman, pooled into.4 ml of distilled water, and immediately cooled in an ice bath. The cells were ruptured with 15 strokes of a microhomogenizer (Micro-Metric Instrument Co.), and a portion of the homogenate was removed for enzyme and protein assays. The remainder was centrifuged at 12 X g for 15 min at 4 C, and the resulting pellet was used for the /8/6648+5$.5/ 198 Assoc. for Res. in Vis. and Ophthal., Inc. Downloaded From: on 11/17/218

2 Volume 19 Number 6 Plasma membrane isolation, corneal endothelial cells 649 isolation of the cell membranes by the aqueous two-phase polymer system developed by Brunette and Till 7 and modified by Lesko et al. 8 The polymer separation solution was prepared by mixing 5 gm of 2% dextran 5 in water (w/v), 26 gm of 3% polyethylene glycol 6 in water (w/v), 25 ml of distilled water, 83 ml of.22m phosphate buffer, ph 6.5, and 2 ml 1~ 2 M ZnCl 2 in a separatory funnel. The top and bottom phases were collected after 48 hr at 4 C. The pellet obtained from the low-speed centrifugation was suspended in 2 ml of the top phase solution, and 2 ml of the bottom phase solution was added and mixed. Centrifugation at 3 x g for 15 min at 4 C resulted in a banding of the membrane material at the interface of the twophase system. This material was removed, transferred to new phase solutions, and rebanded twice. The membrane fraction was then collected and washed three times with distilled water by centrifuging at 3 X g for 15 min in the cold. Electron microscopy. Membrane material from the interface was removed and fixed overnight at 4 C with 2.5% glutaraldehyde buffered with Millonig. After being rinsed with buffer, the material was postfixed in 1% osmium tetroxide for 1 hr and dehydrated with ethanol followed by two changes of propylene oxide. The membranes were then embedded in Epon 812, sectioned, and stained with uranyl acetate and lead citrate. Photomicroscopy was done with a Zeiss EM 9S2 electron microscope. Enzyme assays. The locations of the enzymes used as markers are illustrated in Fig. 1. Mg ++ activated ATPase activity was measured by the method of Bonting et al. 9 and recently adapted to the study of rabbit endothelial homogenates. 4 The activity of Na + -K + - dependent ATPase was determined by measuring the increase in inorganic phosphorus released when potassium is added to a substrate medium already containing magnesium and sodium. This activity could be abolished by the addition of 1 mm ouabain. Each assay was performed in duplicate using 1 /x.1 aliquots of the membrane fraction or of the crude homogenate in a total reaction volume of.1 ml and included controls for endogenous phosphorus and nonenzymatic release. The activity of 5'-nucleotidase was determined by measuring the release of inorganic phosphorus from 5'-AMP (Sigma Chemical Co., St. Louis, Mo.) after 3 min of incubation. 1 Assays were performed on four subcellular fractions: the starting homogenate, the supernatant from the first low- Na-K-ATPase, 5'-Nucleotidase Glucose-6-Phosphatase Fig. 1. Location of the enzyme markers. A 7, Nucleus; M, mitochondria, E.R., endoplasmic reticulum. speed centrifugation, the pellet from the first twophase centrifugation, and the membrane fraction. Glucose-6-phosphatase was assayed in all four subcellular fractions by the method of Hubscher and West" with.1m sodium acetate buffer, ph 6.1, and an incubation time of 15 min. Protein was measured fluorometrically by labeling 1 /xl aliquots of each fraction with fluorescamine (Sigma) at a ph of 9.5 and compared to a standard curve of bovine serum albumin. 12 Results Electron microscopy. Fig. 2 shows an electron micrograph of the cell membrane material found at the interface of the aqueous two-phase system. The membranes appeared mainly as vesicles ranging from.5 to 3 fxm in diameter. No other cellular organelles were apparent. A higher magnification of the same preparation (Fig. 2, inset) shows the bilayer structure of the isolated membranes. Enzyme assays. All enzyme assays were performed on the day of isolation. The results of the ATPase assays performed on the homogenate and membrane fractions of two separate preparations are shown in Table I. Mg ++ -ATPase activity in the membrane fractions of both preparations was approximately twice that of the homogenate; this is consistent with the fact that the enzyme is also located on the cell membrane. The specific activity of the plasma membrane marker Na + -K + -ATPase increased in one preparation from.21 nm//xg/hr in the homogenate to 1.79 nm//xg/hr in the membrane fraction representing a 8.5-fold enrichment for membrane marker. In the sec- Downloaded From: on 11/17/218

3 Invest. Ophthalmol. Vis. Sci. June Zam, Cerda, and Polack Fig. 2. Electron micrograph of the membrane fraction, (x38,4.) Inset, Different field at a higher magnification, (x 18,.) ond preparation the activity increased from.91 to 6.62 nm//-ig/hr, a 7,3-fold enrichment, After correction for that portion of total homogenate removed before the isolation procedure, the membrane fractions contained 41% and 49% of the specific activity of the enzyme marker and 5,5% of the homogenate protein (Table I). Table II shows the assays performed on all four subcellular fractions of two additional preparations. 5'-Nucleotidase, a standard plasma membrane marker, was found to be present in the supernatant fractions of both preparations but with slightly less activity than that found in the homogenate. However, the specific activities of this enzyme in the membrane fractions were 6.6 and 4.7 times those of the starting homogenates, with recoveries of 26.4% and 17.6%. Glucose-6-phosphatase was chosen as the marker enzyme for smooth endoplasmic reticulum. The recovery of this marker was restricted to the supernatant fraction, which contained 62.5% and 83.6% of the specific activity. The activity of this enzyme in the membrane fraction was below the level of detection by the assay (6 x 1~4 U). In these two preparations the purified membranes accounted for approximately 4% of the total homogenate protein, with the majority recovered in the supernatant fractions. Discussion The endothelium consists of a single layer of cells 2 to 3 jam thick lining the posterior surface of the cornea, and although it represents less than 1% of that tissue, it is of para- Downloaded From: on 11/17/218

4 Volume 19 Number 6 Plasma membrane isolation, corneal endothelial cells 651 Table I. Specific activities of Mg-ATPase and Na-K-ATPase in membrane and homogenate fractions \A(/++ ATP/i Qi? (nm/ng/hr) nm/fig/kr Na + -K + ATPase Mg Protein Homogenate: Membrane: * t Degree of membrane purification: *8.5X; t7.3x. Table II. Specific activities of 5' nucleotidase and glucose-6-phosphatase in different subcellular fractions Homogenate: Supernatant: Pellet: Membrane: * t nm/fag/3 min 'nucleotidase Degree of membrane purification: *6.6X; t4.7x Glu cose-6-phospha tase nm/ixg/3 min Mg Protein mount importance for the normal functioning of the cornea. Direct biochemical analysis of the corneal endothelium has been difficult due to the paucity of the material. We have adapted an aqueous two-phase polymer system for the isolation of the plasma membranes from rabbit endothelial cells. This procedure, which separates plasma membranes on the basis of their surface properties, allows the preparation of purified membranes and enzymatic assays to be completed on the same day, a major advantage if the method is to be applied to the study of human corneas. In addition, the aqueous two-phase system requires only low-speed centrifugations for short periods of time, thereby reducing the possibility of contamination by other cell constituents. The enrichment in the membrane fraction for two marker enzymes, Na + -K + -ATPase and 5'-nucleotidase, indicates a high concentration of plasma membrane. The most likely contaminant in this isolation procedure, smooth endoplasmic reticulum, was found to be absent from the membrane fraction by assay for the marker enzyme glucose-6- phosphatase. However, the possibility of minimal contamination by other cell organelles cannot be excluded. That some loss of material occurs, most likely within the two-phase solutions, is shown by the percentage recoveries of the enzyme markers. However, the exceedingly good recovery of Na + -K + -ATPase indicates a high yield of membrane material. This is the first report of cell membrane isolation and direct determination of enzymatic activity in rabbit corneal endothelial Downloaded From: on 11/17/218

5 652 Zam, Cerda, and Polack Invest. Ophthalmol. Vis. Sci. June 198 cells. Membrane-bound enzymes play an important role in fluid and electrolyte transport, 13 ~ 16 and it would be of value to determine their activity in normal human corneas first and then in corneas with diseased endothelia that may require transplantation. However, the techniques must be refined to be able to repeat these studies in small corneal buttons. Experiments are underway to characterize further the plasma membrane of rabbit, dog, and human corneal endothelial cells. Mr. John Valenti performed the electron microscopy. REFERENCES 1. Baum JL: Histochemical study of corneal respiratory enzymes. Arch Ophthalmol 7:59, Kaufman HE, Capella J, and Robbins J: Study of enzyme activity in corneal repair. INVEST OPHTHAL- MOL 3:35, Pena J and Polack FM: Histochemical changes in endothelium of corneas stored in moist chambers. Arch Ophthalmol 72:811, Anderson El, Fischbarg J, and Spector A: Fluid transport, ATP level and ATPase activities in isolated rabbit corneal endothelium. Biochim Biophys Acta 37:557, Riley MV et al: Elimination of anions derived from glucose metabolism as substrates for the fluid pump of rabbit endothelium. Exp Eye Res 24:255, Riley MV, Hodson SA, and Orr HT: Synthetic activity of corneal endothelium. Isr J Med Sci 8:1519, Brunette DM and Till JE: A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J Membr Biol 5:215, Lesko L et al: A rapid method for the isolation of rat liver plasma membranes using an aqueous twophase polymer system. Biochim Biophys Acta 311: 173, Bonting SL, Simon KA, and Hawkins NM: Studies on sodium-potassium-activated adenosine triphosphatase. Arch Biochem Biophys 95:416, Widnell CC and Unkeless JC: Partial purification of a lipoprotein with 5'nucleotidase activity from membranes of rat liver cells. Proc Natl Acad Sci USA 61:15, Hubscher G and West GR: Specific assays of some phosphatases in subcellular fraction of small intestinal mucosa. Nature 25:799, Undenfriend S et al: Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178: 871, Trenberth SM and Mishima S: The effect of ouabain on the rabbit corneal endothelium. INVEST OPH- THALMOL 7:44, Takahasi GH: The relation of corneal anoxia to corneal hydration. INVEST OPHTHALMOL 6:562, Mishima S and Kudo T: The in vitro incubation of rabbit cornea. INVEST OPHTHALMOL 6:329, Dikstein S and Maurice D: The metabolic basis to the fluid pump in the cornea. J Physiol (Lond) 221:29, Downloaded From: on 11/17/218