THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 8, Issue of April 25, pp. 2041-2048, 1969

Ptinted in U.S.A.

The Lens Capsule

SUGAR AND AMINO ACID COMPOSITION*

(Received for publication, November 29, 1968)

SUGURU FUKUSHI~ AND ROBERT G. SPIRO

From the Departments of Biological Chemistry and Medicine, Harvard Medical School, the Elliott P. Joslin Re- search Laboratory, and the Peter Bent Brigham Hospital, Boston, Massachusetts 02215

SUMMARY

Anterior and posterior portions of the lens capsules of calf and cow eyes were obtained free of cellular material by a procedure which included ultrasonic treatment.

Sugar analyses indicated that approximately 11% of the dry weight of the capsules was in the form of monosaccharide residues, which were identified as glucose, galactose, man- nose, fucose, hexosamines, and sialic acids. Small amounts of hexuronic acids were also present. The hexosamines consisted of glucosamine and galactosamine, which were present in the ratios of 92:8 in the calf anterior capsule and 93 : 7 in the calf posterior capsule. The sialic acids consisted of both N-glycolyl- and N-acetylneuraminic acids in the ratio of 60:40 in the anterior calf capsule and 65 :35 in the posterior calf capsule.

Distinct differences with age were noted in the composition of the lens capsule. Cow capsules contained larger amounts of glucose and galactose, and smaller levels of mannose and hexosamine than those from calves. Decreases in the sialic acid content were observed in the older animals while the fucose remained constant with age.

The posterior portions of the capsules of both age groups contained less glucose and galactose than the anterior parts, but more sialic acid and hexuronic acid.

The peptide portion of the capsules was characterized by the occurrence of a large amount of glycine (approximately one-fourth of the amino acid residues), as well as of hydroxy- proline (10% of the amino acid residues) and hydroxylysine (4% of the amino acid residues). In addition, all of the com- monly occurring amino acids were present, including cystine, methionine and tryptophan.

Amide analyses indicated that nearly half of the dicar- boxylic amino acids were in the uncharged form; in the anterior calf capsule 45% of these ammo acids were in the amide form. An accounting of the charged groups of the capsules indicated that each had a net positive charge; the

* This work was supported by Grants AM 10482 and HE 11306 from the National Institutes of Health.

$ Present address, Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan.

anterior calf capsule had a positive charge of 25 per 1000 amino acid residues.

Of particular note in the amino acid composition of the lens capsules was the occurrence of very high hydroxylyslne to lysine and hydroxyproline to proline ratios, which increased still further with age.

While the composition of these lens capsules place them in the collagen group of proteins, they appear most closely related to other extracellular membranes, such as the renal glomerular basement membrane.

The lens of the eye is completely enclosed by a membrane called the lens capsule which is believed to be a secreted product of epithelial cells (1) and which may play an important regulatory function in controlling the internal environment of the lens. The thicker anterior portion of this capsule rests on a layer of these epithelial cells, while the thinner posterior portion, after embryonal development, is in direct contact with the lens. Although this membrane does not have a fibrillar structure under the electron microscope, its x-ray diffraction pattern and its content of hydroxyproline indicate that it is related to the collagen group of proteins (2).

Immunochemical studies have shown that the lens capsule is related to the renal glomerular basement membrane (3), as well as to the basement membranes found in a large number of other mammalian tissues (4). These basement membranes are also related by their high carbohydrate content, which is re- sponsible for their intense staining with the periodic acid- Schiff reagent (5). It has been known for some time that the lens capsule of the eye contains approximately 10% carbohy- drate, and the presence of neutral sugars, hexosamine, sialic acid, and hexuronic acid has been reported (2, 6-8).

Because of the relative ease of isolation of the lens capsule, it presents itself as an attractive subject for elucidating the struc- tural characteristics common to the large group of extracellular membranes known as basement membranes.

In the present study detailed analyses of the sugar and amino

2041

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Composition of Lens Capsule Vol. 244, No. 8

acid composition of both the anterior and posterior portions of the lens capsule are reported. Analyses are given for capsules isolated from calves and cows in order to evaluate the effect of aging on this membrane.

EXPERIMENTAL PROCEDURE

Preparation of Lens Capsules-Eyes from freshly killed calves (approximately 3 weeks of age) and of cows were brought on ice to the laboratory and were immediately dissected. After removal of the lens from the eye, it was rolled on glass fiber paper wetted with 0.85% NaCl to remove any adhering vitreous body or iris pigment. The lens capsule was divided into anterior and posterior portions by cutting with scissors 2 to 3 mm on each side of the equator so as to avoid contamination with the zonular fibers which are attached at the equator. The. anterior and posterior capsules were washed separately at 2’ with 0.85% NaCl (3 ml per capsule) with rapid shaking in order to remove attached epithelial cells and vitreous. Three changes of the sodium chloride solution were used over a total period of 42 hours, and each time the lens capsules were separated from the wash by centrifugation in a refrigerated centrifuge.

In order to remove any remaining cells, the washed lens capsules were treated by ultrasonic disruption, by using aBranson Sonifier (model S-125), with a probe terminating in a 0.5-inch radiating area. For this purpose, approximately 80 lens capsules were suspended in 20 ml of 0.85% NaCl in a 50-ml beaker placed in an ice bath. Sonic disruption was carried out at a No. 4 power setting for intervals of 30 see, followed by cooling to 2”. The total sonic treatment for the anterior capsule was 3 min while that for the posterior capsule was 30 sec.

The sonically treated capsules were centrifuged at 3000 rpm at 2” for 20 min and were then washed four times with large volumes of ice-cold distilled water in order to remove the sodium chloride. The capsules were then lyophilized and stored in a desiccator at -20”. Prior to each weighing, the capsules were dried to constant weight in vacuum at room temperature with a phosphorus pentoxide trap., The dry weight of the capsules was determined by heating at 80” in a vacuum oven; under these conditions an additional 4 to 6% of the capsule weight was lost.

Analysis of Sugars-For the release of neutral sugars, the capsules were hydrolyzed in 2 N sulfuric acid for 4 hours at 100” in sealed tubes. The hydrolysate was passed through coupled Dowex 50 and Dowex 1 columns (9). The neutral sugars were identified in the solvent systems previously employed (10). For the determination of glucose, galactose, and mannose, quantita- tive chromatography on Whatman No. 1 paper in l-butanol- ethanol-water (10:1:2) was used (11). In this method, the sugars eluted from the chromatogram were determined by the ferricyanide reaction, while the glucose alone was measured with the use of glucose oxidase.

Total neutral sugars were determined by the anthrone reagent (9). Fucose was determined by the method of Dische and Shettles (12) with a 3-min heating period.

The hexosamines were analyzed after hydrolysis of the capsules in 4 N HCl at 100” for 6 hours in sealed tubes (9). After adsorp- tion and elution from small Dowex 50 columns, they were determined by the Elson-Morgan reaction (13). The hexos- amines were identified either by direct chromatography in pyridine-ethyl acetate-water-acetic acid (5: 5 : 3 : 1) (14) or by chromatography of their pentose derivatives in l-butanol- ethanol-water (4 : 1: 1) after the ninhydrin degradation (9). The glucosamine and galactosamine were further identified and

estimated by chromatography on the Technicon amino acid analyzer after they had been freed of most amino acids by N-acetylation, passage through Dowex 50 and Dowex 1 resins, and deacetylation (11).

Sialic acid was determined by the thiobarbituric acid assay after hydrolysis of the lens capsule in 0.1 N sulfuric acid at 80” for 60 min (15). For the purpose of identification, the 0.1 N

sulfuric acid hydrolysate was passed through Dowex 1, and the sialic acids were eluted with 0.3 N formic acid and chromato- graphed in n-butyl acetate-acetic acid-water (3 : 2: 1) (9). N-Glycolylneuraminic acid was estimated by measuring the glycolic acid content of the sialic acid fraction (9)

Hexuronic acid analyses were performed by the Dische carba- zole procedure (16), with glucuronic acid as a standard. Sepa- rate galactose, glucose, and mannose standards were also run with these analyses to correct for the contribution of these sugars to this reaction.

Analysis of Peptick Portion-For the determination of the amino acid content of the lens capsules, hydrolysis was performed in glass-distilled constant boiling HCl in sealed tubes at 105” under an atmosphere of nitrogen after repeated evacuation with a vacuum pump and flushing with nitrogen. Hydrolysis was carried out for periods of 26 to 72 hours at a concentration of 1 mg of capsule per ml.

The cystine and cysteine content of the capsule was de- termined as cysteic acid after oxidation of the capsule with performic acid at 0” for 20 hours by the method of Schram, Moore, and Bigwood (17). The oxidized protein was hydrolyzed at 105” for 24 hours.

The amino acid analyses were performed on aliquots of the hydrolysates with the use of the Technicon ammo acid analyzer.

For the determination of tryptophan, the capsules were hydrolyzed with 4 N Ba(OH)t for 70 hours at 110’ (18). The hydrolyses were carried out in polypropylene tubes (10 x 100 mm) placed inside Kimax test tubes (18 x 150 mm) which were then sealed under nitrogen, after repeated evacuation with a vacuum pump and flushing with nitrogen. After neutralization with CO2 and washing of the barium carbonate precipitate, an aliquot of the hydrolysate was analyzed with the Technicon amino acid analyzer with the buffer modification previously described (11). The tryptophan was corrected for adsorptive losses on the basis of the leucine content of the sample (18).

The amide content was determined after hydrolysis of the capsule in 2 N HCl at 100” for 3 hours, by a modification of the Conway microdiffusion technique which eliminates any contribu- tion of ammonia from the hexosamines (19).

Other Chemical Analyses-Total nitrogen was determined by the micro-Kjeldahl procedure of Koch and McKeekin (20). Sulfate esters were determined by the benzidine method of Dodgson and Spencer (21), after hydrolysis in 4 N HCl at 100” for 22 hours in sealed tubes and passage of the hydrolysate through columns of Dowex 50 as previously described (11). Total phosphorus was analyzed by the ascorbic acid method of Chen, Toribara, and Warner (22) after digestion with 70% perchloric acid (23).

DNA analyses were made by the diphenylamine reaction of Burton (24) after hydrolysis of the capsules in 0.5 N perchloric acid at 90” for 17 min. For the determination of RNA, the capsules were hydrolyzed in 0.1 N KOH at 37” for 16 hours (25). The RNA content was determined by the orcinol reaction for ribose on the perchloric acid supernatant of this hydrolysate (26). In addition to ribose, glucose, galactose, mannose, and fucose

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1969 8. Fukushi and R. G. Spiro 2043

standards were analyzed in order to permit correction for the contribution of these hexoses, solubilized by the alkali treatment, to the orcinol reaction.

RESULTS

Preparation of Lens Capsules-The capsule preparations were examined under the phase microscope and after staining with hematoxylin. Although some epithelial cells adherent to the anterior lens capsule were observed after the shaking procedure, these cells were completely eliminated by the sonic treatment (Fig. 1).

DNA analyses indicated the absence of this component (less than 0.1%). RNA determinations showed that the capsules were also free of this nucleic acid.

Total phosphorus analyses on several preparations indicated

FIG. 1, Photomicrograph of isolated anterior calf lens cap- sule. X 9.5.

TABLE II

Carbohydrate composition of anterior and posterior parts of cow lens capsule

Monosaccharide

Percentage of dry weight of capsulea Micromoles

per 100 mg of capsule

Anterior Posterior Ante- Poste- rior rior

--

Glucose. 5.30 f 0.05 (16) 4.41 f 0.11 (17) 29.4 24.5 Galactose.. . 5.45 i 0.14 (16) 5.05 i 0.13 (15) 30.3 28.0 Mannose. . . 0.51 * 0.020 (9) 0.55 i 0.018 (8) 2.8 3.1 Fucose.. 0.25 f 0.017 (9) 0.28 i 0.13 (9) 1.5 1.7 Hexosaminesb.. 0.72 f 0.016 (16) 0.96 f 0.020 (6) 4.0 514 Sialic acidsc. . 0.11 f 0.003 (3) 0.23 f 0.007 (3) 0.3 0.7 Hexuronic

acids. . . . . 0.06 f 0.007 (4) 0.22 i 0.018 (3) 0.3 1.1

(L Values are expressed as the mean & S.D. The figures in parentheses represent the number of samples analyzed.

b Expressed as free base; if expressed in the N-acetyl form the total hexosamine would be 0.89yo for the anterior capsule and 1.18% for the posterior capsule; over 95% of the hexosamine was present as glucosamine.

c Expressed as N-acetylneuraminic acid; both N-acetyl- neuraminic acid and N-glycolylneuraminic acid were identified in the sialic acid fraction.

an average phosphorus content of 0.008% for the calf anterior capsule; 0.032Yo for the calf posterior capsule; 0.006~o for the cow anterior capsule; and 0.014yo for the cow posterior lens capsule.

The isolated lens capsule stained intensely with the periodic acid-Schiff reagent. The average weights of the isolated ly- ophilized capsules were as follows: 0.48 mg per calf anterior capsule; 0.27 mg per calf posterior capsule; 4.2 mg per cow anterior capsule; and 1.1 mg per cow posterior capsule.

Carbohydrate Composition-Glucose, galactose, mannose, and fucose were identified as the neutral sugar components of the lens capsule by paper chromatography. The quantities of these sugars in the calf anterior and posterior and cow anterior and posterior capsules are shown in Tables I and II. It may be noted that the glucose and galactose content of the cow capsule

TABLE I Carbohydrate composition of anterior and posterior parts of calf lens cqsule

Monosaccharide

Glucose ........................... Galactose .......................... Mannose. ......................... Fucose ............................ Hexosaminesb. .....................

Glucosamine ..................... Galactosamine ..................

Sialic acids ........................ N-Glycolylneuraminic acid ....... N-Acet,ylneuraminic acid .........

Hexuronic acid. ...................

-

- I Percentage of dry weight of capsule0

Anterior Posterior Anterior Posterior

4.08 f 0.13 (28) 4.75 i 0.24 (22) 0.74 f 0.033 (24) 0.25 i 0.012 (12) 1.60 f 0.03 (8) 1.47 0.13 0.31 f 0.007 (12) 0.18 0.13 0.33 f 0.07 (5)

3.22 i 0.07 (10) 3.72 f 0.22 (9) 0.71 f 0.043 (20) 0.24 i 0.007 (11) 2.27 i 0.04 (8) 2.11 0.16 0.65 f 0.026 (12) 0.41 0.23 1.29 i 0.13 (5) I I

22.7 17.9 26.3 20.7

4.1 3.9 1.5 1.5 8.9 12.7 8.2 11.8 0.7 0.9 1.0 2.0 0.6 1.3 0.4 0.7 1.7 6.7

Micromoles per 100 mg of capsule

= Values are expressed as the mean i SD. The figures in parentheses represent the number of samples analyzed. b Expressed as free base; if expressed in the N-acetyl form the total hexosamine would be 1.97yo for the anterior capsule and 2.80%

for the posterior capsule.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

is significantly higher than that of the calf, and that the anterior amino acids are presented in Tables III and IV. Complete capsule at both ages has a larger amount of these two sugars release of all of the amino acids except isoleucine and valine than the posterior portion of the capsule. In contrast, the occurred by 26 hours. A small destruction of serine and threo- level of mannose is higher in the younger animals, and there is nine occurred, which was corrected for by extrapolation to zero no apparent difference in the amount of this sugar present in time by the method of least squares. the anterior and posterior portions of the capsules. The fucose The cow lens capsules were analyzed under identical conditions. content does not appear to differ with age or from one portion Timed acid hydrolysis resulted in a similar rate of release and of the capsule to the other. destruction of the various amino acids. The average or extra-

Analyses of the neutral sugar fraction performed by the an- polated values for the amino acids from the cow capsules are throne reaction on the unhydrolyzed capsules gave the following shown in Table IV. values, expressed as glucose equivalents per dry weight of the Of note in these amino acid analyses of the capsules from both membrane: calf anterior capsule, 7.92 f 0.07%; calf posterior, age groups is the presence of a large number of glycine residues 6.74 f 0.17%; cow anterior capsule, 9.55 f 0.21%; and cow which make up approximately 25oJ, of the total amino acid posterior, 8.36 f 0.22%. The sums of the neutral sugars residues. The occurrence of large amounts of hydroxyproline galactose, mannose, glucose, and fucose, as determined indi- and hydroxylysine can also be seen. These three amino acids vidually after hydrolysis, when expressed as glucose equivalents are present in larger amounts in the cow capsules than in the after correction for the diierence in color yields given by these calf, and in the anterior portions of the capsules of both age sugars in the anthrone reaction (9), were in good agreement with groups. the anthrone determinations, with the following calculated The amide analyses indicated that approximately 45% of the values: anterior calf, 7.75%; posterior calf, 6.01%; anterior cow, dicarboxylic acid residues of the calf capsules and 48% of the 9.05%; and posterior cow capsule, S.OO~o. In making these dicarboxylic acids of the cow capsules were present in the amide calculations, the very small contributions which the sialic acid form (Table IV). and the glucuronic acid make to the anthrone reaction were also Xolubilixation of Capsules with Dilute Allcali-Treatment of the taken into account. calf and cow lens capsules with 0.1 N NaOH at 37” (10 mg of

The hexosamine fraction consisted primarily of glucosamine, capsule per ml) caused the solubilization of more than 90% of the with a small amount of galactosamine being present in each of peptide and carbohydrate portions of the capsules. This was the capsules. The ratio of glucosamine to galactosamine, as determined by analysis for the various sugar components and determined by ion exchange chromatography, was 92:8 in the amino acids on the solubilized material. Approximately equal calf anterior capsule while it was 93:7 in the calf posterior cap- solubilization of all of the sugar and amino acid components sule. These sugars were further identified by direct chromatog- took place by this treatment. raphy in the Fischer-Nebel system and by the formation of their pentose derivatives after ninhydrin degradation. DISCUSSION

The levels of hexosamine in the calf capsules were higher than The lens capsules used in the present study were obtained those in the cow, and the posterior portions of the capsules con- essentially free of cellular material, as determined from morpho- tained more hexosamines than the anterior portions. The in- logical examination, as well as from analyses for DNA, RNA, creased amounts present in the posterior capsules are most and phosphorus. probably due to the higher levels of mucopolysaccharides as The total sugar residue weight accounted for approximately indicated by the hexuronic acid contents of these portions. 11% of the dry weight of the capsules (Table IV). This repre- When the level of hexosamine is adjusted by subtracting an sents approximately 85 sugar residues per 1000 amino acid amount equivalent to the hexuronic acid present, similar levels residues. The major sugar components present were glucose of hexosamines are obtained for the anterior and posterior parts and galactose. The content of these two sugars increased with of the capsules, although the calf capsules still contain substan- age and they were present in larger amounts in the anterior than tially more hexosamine than those of the cow. in the posterior portions of the capsules. The content of man-

Paper chromatography of the sialic acid fraction from both the nose and non-mucopolysaccharide hexosamine on the other hand calf and cow capsules showed the presence of both N-glycolyl- decreased with age, but was approximately equal in the anterior and N-acetylneuraminic acid. In the calf capsule the molar and posterior portions of the capsule. While similar levels of ratio of N-glycolyl- to N-acetyhreuraminic acid was 60 : 40 in the fucose were found in calf and cow capsules, a pronounced de- anterior portion and 65:35 in the posterior part. The levels of crease in the sialic acid content occurred with age, so that the the sialic acids were considerably higher in the calf capsules than total number of residues present in the form of these two ter- in the cow and in the posterior portions of the capsules than in minally located sugars manifested a decline with age. The the anterior part. sialic acids were present in larger amounts in the posterior cap-

Hexuronic acid levels, expressed as glucuronic acid, were again sules than in the anterior portions, while the fucose levels did higher in the calf than in the older animals, and substantially not differ between these two portions of the capsules. greater in the posterior part of the capsule than in the anterior All of the capsules showed the presence of both the N-glycolyl portion. and N-acetyl forms of neuraminic acid, with a predominance of

Sulfate analyses gave the following results, expressed per dry the former. N-Glycolylneuraminic acid is usually the major weight of the capsule: anterior calf, 0.46%; posterior calf, 0.59’%; form of sialic acid in bovine tissues (27), and similar ratios of anterior cow, 0.21%; and posterior cow, 0.27%. N-glycolyl- to N-acetylneuraminic acid have been found in the

Amino Acid Composition-Table III gives the amino acid bovine renal glomerular basement membrane (11). values obtained after acid hydrolysis of the calf capsules for 26 Over 90% of the hexosamines in both portions of the lens to 72 hours. The average or extrapolated values for the various caosule at both aces were identified as alucosamine. with nalac- .._ .~ -~

2044 Composition of Lens Capsule Vol. 244, No. 8

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1969 S. Fukushi and R. G. Spiro 2045

TABLE III

Amino acid composition of anterior and posterior parts of calf lens capsule

Amino acid

Amount in anterior capsule after hydrolysis for

26 hrs (2)b 48 hrs (2) 72 hrs (2)

Average or extrap- elated values

Amount in posterior capsule after hydrolysis for Average or &rap

&ted value” 26 hrs (2) 48 hrs (2) 72 hrs (1)

&w?w1cs/100 nag, dry wt

Hydroxyproline.. . . . . . 76.5 72.0 74.9 74.5 Aspartio acid.. . . . . . . 45.9 46.1 44.9 45.6 Threonine................. 28.4 27.3 26.7 29.30 Serine..................... 41.6 39.1 36.1 44.8~ Glutamic acid. . . . . . . 74.6 72.1 72.5 73.1 Proline. . . . . . . . . . . . 52.4 50.5 54.9 52.6 Glycine. . . . . . . . . . 192.8 186.1 184.1 187.7 Alanine.................... 36.9 37.0 35.6 36.5 Valine..................... 26.4 27.3 28.5 27.9d Methionine................ 10.9 11.3 11.0 11.1 Isoleucine . . . . . . 21.5 23.9 23.7 23.gd Lencine . . . . . . . . . . . . . . 46.7 46.5 48.0 47.1 Tyrosine . . . . . . . 10.8 11.5 10.8 11.0 Phenylalanine . . . . . . . 22.3 24.1 23.1 23.2 Hydroxylysine.. . . . . . . 28.4 30.0 29.3 29.2 Lysine..................... 10.6 12.1 10.8 11.2 Histidine. . . . . . . 12.4 14.1 13.4 13.3 Arginine................... 31.1 33.3 32.6 32.3 Half-cystine. . . . . 19.3e Tryptophan. . . . . 3.4’ Amide nitrogen.. . 53.30

a Unless otherwise indicated these values represent an average of values obtained at 26,48, and 72 hours.

* Figures in parentheses indicate number of analyses for each time; average values for each time are given.

d Average of values obtained at 48 and 72 hours. e Determined as cysteic acid after performic acid oxidation. f Determined after alkaline hydrolysis. 0 Average of three determinations by microdiffusion.

c These values are extrapolated to zero hydrolysis time.

tosamine making up the remainder. This is again similar to the proportion in which these two amino sugars occur in the bovine renal glomerular basement membrane (11).

The occurrence of hexuronic acid indicates the presence of some mucopolysaccharides, primarily in the posterior part of the calf lens capsule. The posterior lens capsules contained not only more hexuronic acid, but also approximately an equiva- lent amount of extra hexosamine when compared to the anterior portion at each age.

The monosaccharides identified in the present investigation of the bovine lens capsule are in agreement with those previously reported (2, 6-S). In addition this study has determined the form in which the sialic acids and hexosamines occur and quanti- tative data for the level of all of the sugar components have been obtained. Amino acid analyses for the lens capsule have not been previously reported, although the occurrence of hydroxy- proline and large amounts of glycine had been observed by paper chromatography (2).

tion, but definite changes with age were apparent (Table IV). In comparing the calf anterior capsule to the cow anterior cap- sule (Table V), it may be noted that a substantial increase in the glycine, hydroxyproline, and hydroxylysine residues occurs with age. No change occurs in the total hydroxyamino acids as a decrease in the levels of serine, threonine, and tyrosine is balanced by an increase in the amounts of hydroxylysine and hydroxy- proline. A marked increase in the ratio of hydroxylysine to lysine occurs with age compared to only a moderate increase in the ratio of hydroxyproline to proline.

All of the capsules have an excess of cationic groups over anionic residues, taking into account the charges present on the basic amino acids, the dicarboxylic amino acids not in the amide form, and the sialic acid residues. The net positive charge per 1000 amino acid residues is as follows: calf anterior capsule 25; calf posterior capsule 27; cow anterior capsule 30; and cow posterior capsule 27.

The analyses of the lens capsules performed in the present study indicate that approximately 80% of the weight is accounted for by the amino acid residues. The amino acids of the lens capsule can be divided into several groups (Table V). The most outstanding characteristic of these analyses is the occur- rence of substantial amounts of hydroxylysine and hydroxy- proline, as well as approximately one-quarter of the total amino acid residues in the form of glycine.

It is evident from the amino acid analyses of the capsule that it can be considered a member of the collagen group of proteins. However, this extracellular membrane differs in important re- spects in its amino acid composition from the fibrous type of collagen which has been so extensively studied from sources such as skin, tendon, and swim bladder (29, 30).

Little difference between the anterior and posterior portions of the lens capsule can be seen in terms of the amino acid composi-

A comparison between the major groups of amino acids of the lens capsule and such a typical collagen (calf skin) (28) and another extracellular membrane (the glomerular basement mem- brane) (11) is presented in Table V. It is apparent that the nonpolar aliphatic amino acids are more abundant in the calf

63.4 44.4 28.1 39.7 69.2 53.1

169.4 35.8 25.0 10.2 22.0 48.5 11.1 21.9 23.5 12.2 13.7 32.8

63.6 62.2 63.1 46.4 46.8 45.9 26.7 26.1 29.10 37.5 36.3 41.3c 71.7 68.5 69.8 53.8 54.4 53.8

166.0 165.5 167.0 36.7 35.9 36.1 27.9 28.5 28.2d

9.5 10.1 9.9 23.4 23.2 23.3d 48.5 47.7 48.2 11.1 10.6 10.9 22.1 21.2 21.7 24.7 25.1 24.4 12.8 13.7 12.9 14.1 14.7 14.2 34.2 33.1 33.4

18.0” 2.4f

52.80

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2046 Composition of Lens Capsule Vol. 244, No. 8

Component

Hydroxyprolineo. ........... Aspartic acid. .............. Threonine. ................. Serine ...................... Glutamic acid. ............. Proline. .................... Glycine ..................... Alanine ..................... Valine ...................... Methionine ................. Isoleucine .................. Leucine .................... Tyrosine ................... Phenylalanine Hydroxylysine .............. Lysine ...................... Histidine. .................. Arginine .................... Half-cystine. ............... Tryptophan. ............... Amide nitrogen”. ...........

Glucosec .................... Galactose ................... Mannose. .................. Fucose ..................... Hexosaminesd. .............. Sialic acids. ................ Hexuronic acids. ...........

Total& ....................

TABLE IV Composition of anterior and posterior parts of calf and cow lens capsule -

-

-

--

Residue weight

Calf I

COW

Anterior Posterior Anterior

g/100 g dry ca~sda

Posterior

8.43 7.14 9.49 9.36 5.25 5.28 4.69 4.88 2.96 2.94 2.17 2.41 3.90 3.60 2.96 3.23 9.44 9.01 8.59 9.08 5.11 5.22 5.23 5.49

10.72 9.54 11.55 10.83 2.60 2.57 2.13 2.35 2.77 2.80 2.23 2.24 1.46 1.30 1.25 1.47 2.69 2.64 2.64 2.67 5.33 5.46 4.90 5.30 1.80 1.78 1.37 1.45 3.42 3.19 3.44 3.34 4.21 3.52 4.89 4.34 1.44 1.65 1.18 1.37 1.82 1.95 1.19 1.54 5.05 5.22 4.23 4.44 1.99 1.86 1.33 1.94 0.63 0.45 0.50 0.47

(0.75) (0.74) (0.72) (0.76)

93.5 83.8 110.7 107.1 57.2 60.9 53.8 54.8 36.8 38.6 28.4 30.8 56.2 54.8 44.9 48.0 91.7 92.6 87.8 90.9 66.0 71.4 71.2 73.0

235.6 221.6 267.1 245.2 45.8 47.9 39.6 42.7 35.0 37.4 29.7 29.2 13.9 13.1 12.5 14.5 29.9 30.9 30.8 30.5 59.1 64.0 57.2 60.5 13.8 14.5 11.1 11.5 29.1 28.8 30.9 29.4 36.7 32.4 44.8 38.9 14.1 17.1 12.1 13.8 16.7 18.8 11.5 14.5 40.5 44.3 35.8 36.7 24.2 23.9 17.0 24.3

(6:::) (7:::) (6;::) (730:;)

3.67 2.90 4.77 3.97 28.5 23.8 38.8 31.7 4.28 3.35 4.91 4.55 33.0 27.5 40.0 36.2 0.67 0.64 0.46 0.50 5.1 5.2 3.7 4.0 0.22 0.21 0.22 0.25 1.9 2.0 2.0 2.2 1.81 2.58 0.82 1.19 11.2 16.9 5.3 7.0 0.29 0.61 0.10 0.22 1.3 2.7 0.4 0.9 0.30 1.17 0.05 0.20 2.1 8.9 0.4 1.4

92.26 88.58 87.29 89.08

0 Amino acid values calculated from average or extrapolated values.

b Parentheses indicate values that are not included in totals. e Monosaccharide values calculated from analyses given in

Tables I and II. d Expressed as N-acetylhexosamine.

d The average total nitrogen contents were as follows: calf anterior capsule 14.1 f 0.2%; calf posterior capsule 14.1 i 0.4%; cow anterior capsule 14.4 f 0.1%. The amino acid, amide, and amino sugar analyses accounted for 100% of the total nitrogen in the calf anterior capsule; 98yo in the calf posterior capsule; and 93yo of the cow anterior capsule.

skin collagen, and this is primarily due to glycine residues. This amino acid makes up approximately one-third of the residues of calf skin collagen, one-fourth of the residues in the lens capsules, and one-fifth of the residues in the glomerular basement mem- brane. On the other hand, the hydroxyamino acids are con- siderably more abundant in the membranes than in the fibrous collagen. There is also a much larger number of aromatic residues in the membranes than in the calf skin collagen, and this is due to the occurrence not only of a larger number of tyrosine and phenylalanine residues, but also to the presence of tryptophan, an amino acid which is generally regarded as being absent in the collagens. The membranes have a larger number of dicarboxylic amino acids, but approximately the same propor- tion of these are present in the amide form as in the collagens.

cow anterior lens capsule has approximately 6 times as many hydroxylysine residues as the calf skin collagen, while the glo- merular basement membrane has about 3 times as many residues of this amino acid as the collagen. The ratio of hydroxylysine to lysine differs substantially among these proteins, being as high as 3.7 in the cow anterior lens capsule and as low as 0.27 in the skin collagen.

The main difference in the basic amino acids is the markedly greater number of hydroxylysine residues present in the mem- branes when compared to the skin collagen. For example, the

The imino acids are present in larger amounts in the skin collagen than in the membranes, and there is a pronounced difference in the ratio of hydroxyproline to proline among these proteins. This ratio is as high as 1.55 in the cow anterior cap- sule and as low as 0.68 in the skin collagen, while it is close to unity in the glomerular basement membrane. It is apparent that a greater degree of hydroxylation of proline as well as of lysine occurs during the synthesis of the membranes compared to the fibrous collagens.

Sulfur-containing amino acids are present in amounts close to

T .-

.-

.-

Residues per 1000 total amino acid residues

Calf

Anterior Posterior Anterior Posterior

i cow

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of April 25, 1969 S. Fukushi and R. G. Spiro 2047

TABLE V

Comparison of amino acid patterns of lens capsule, glomerular basement membrane, and calf shin collagen

Amino acid

Nonpolar aliphatic.. Glycine. . .

Imino acids................. Hydroxyproline. . . . . (Hydroxyproline: proline)”

Hydroxyamino acidsd. . Acidic amino acids.. .

Amide groups.. . . Free carboxyl groups. .

Basic amino acids. . . Hydroxylysine. . . . (Hydroxylysine:lysine)c..

Aromatic amino acids.. . Sulfur-containing amino

acids................... -

anterior letI.

capsule

405 236 160

(1.9442) 237 149

67 82

108

(2.3671) 47

424 267 182 111

(1.55) 240 142

68 74

104

(3.4751) 46

395 488 208 320 137 232

(0.:) (0.9648) 201 158 164 117

68 46 96 71

115 89

(0.2823) (Z7) 52 16

38 30 45 4.3

a Data taken from Spiro (11). b Data taken from Pies and Gross (28). c Figures in parentheses represent molar ratios. d Includes serine and threonine as well as tyrosine, hydroxy-

lysine, and hydroxyproline.

10 times as great in the membranes as in the skin collagen. This is due to the larger number of methionine residues in the mem- branes as well as to the occurrence of substantial numbers of half-cystine residues. This latter amino acid has been reported to be absent from the typical vertebrate collagens (29,30).

It is evident from these comparisons that the membranes con- tain a higher number of polar amino acid residues than the fibrous collagens, while a characteristic amino acid such as glycine occurs in smaller amounts. Although the lower levels of glycine would tend to diminish the relationship between the membranes and more typical collagens, the occurrence of large amounts of amino acids characteristic of collagens, namely, hydroxylysine and hydroxyproline, tend to reinforce the concept that these mem- branes are members of the collagen group and that quite diverse ratios of the various amino acid constituents are consistent with such a classification.

It is also of interest to compare the membranes and the col- lagens in regard to their carbohydrate composition. Both the lens capsules and the glomerular basement membrane have approximately 10% of their weight in the form of sugar residues, while most of the fibrous collagens from vertebrate sources con- tain less than 2% total carbohydrate (29, 30). Glucose and galactose are the predominating sugars in both the collagens and membranes, and it has been possible to show that all of the glucose and most of the galactose are present as hydroxylysine- linked carbohydrate units (31-33). The lens capsule and the glomerular basement membrane (11) contain in addition to these two sugars, substantial amounts of mannose, fucose, sialic acids, and hexosamines.

It is apparent that there is a group of extracellular membranes which are related to the collagens by their amino acid composition and by x-ray diffraction, but which do not have the organization of fibrillar collagens as observed under the electron microscope.

These membranes, exemplified by the lens capsule, the glomerular basement membrane, Reichert’s membrane of the yolk sac (34), and the Descemet’s membrane of the cornea (35) tend to have substantially higher amounts of carbohydrate than the collagens, as well as larger amounts of the polar amino acids. They stain deeply with the periodic acid-Schiff reagent.

The occurrence of greater numbers of polar amino acids in the basement membranes than in the fibrous collagens could reflect the occurrence in the membranes of more than one type of sub- unit. On the other hand, it has been shown that skin tropo- collagen has peptide regions with amino acid compositions which are polar in nature and differ substantially from the helical por- tions of the peptide chain (36, 37). By analogy, this group of membranes which belong to the collagen family may also contain such polar regions, but the proportions of these may be greater than is found in the more classical collagens.

The large amount of carbohydrate present in these basement membranes may play an important function in their organiza- tion. In order to evaluate this role it is necessary to understand the structure of this carbohydrate and its relationship to the peptide portion. The results of studies conducted on the lens capsule in order to determine the nature of the carbohydrate units in this membrane will be reported in a succeeding paper (33) *

REFERENCES

1. PIRIE, A., AND VAN HEYNINOEN, R., Biochemistry of the eye, Blackwell Scientific Publications, Ltd., Oxford, 1956, p. 30.

2. PIRIE, A., Biochem. J., 48, 368 (1951). 3. ROBERTS, D. ST. C., Brit. J. Ophthalmol., 41, 338 (1957). 4. CRUICKSHANK, B., AND HILL, A. G. S., .I. Pathol. Bacterial., 66,

283 (1953). 5. LILLIE, R. D., Lab. Invest., 1,30 (1952). 6. GLEGG, R. E., EIDINGER, D., AND LEBLOND, C. P., Science,

120. 839 (1954). 7. DISC&, Z.,‘ AND BORENFREUND, E., Amer. J. Ophthalmol., 33,

165 (1954). 8. DISCHE, Z., ZELMANIS, G., AND ROTHSCHILD, C., Arch. Bio-

them. Biophw.. 121. 685 (19677). 9. SPIRO, R. G:, i”n &. F..NEUFELD AND V. GINSBURG (Editors),

Methods in enzymology, Vol. VIII, Academic Press, New York, 1966, p. 3.

10. SPIRO, R. G., AND SPIRO, M. J., J. Biol. Chem., 240,997 (1965). 11. SPIRO, R. G., J. Biol. Chem., 242, 1915 (1967). 12. DISCHE, Z., AND SHETTLES, L. B., J. Biol. Chem., 176, 595

(1948). 13. BOAS, N. F., J. Biol. Chem., 204, 553 (1953). 14. FISCHER, F. G., AND NEBEL, H. J., Hoppe-Seyler’s 2. Physiol.

Chem., 302, 10 (1955). 15. WARREN, L., J. Biol. Chem., 234, 1971 (1969). 16. DISCHE, Z., J. Biol. Chem.. 167. 189 (1947). 17. SCHRA~, E:, MOORE, S., AND BIGWOOD, E: J., Biochem. J., 67,

33 (1954). 18. NOLTMANN, E. A., MAHOWALD, T. A., AND KUBY, S. A., J.

Biol. Chem., 237, 1146 (1962). 19. SPIRO, M. J., AND SPIRO, R. G., J. Biol. Chem., 237,1507 (1962). 20. KOCH, F. C., AND MCKEEKIN, T. L., J. Amer. Chem. Sot., 46,

2066 (1924). 21. DODGSON, K. S., AND SPENCER, B., Biochem. J., 66,436 (1953). 22. CHEN, P. S., JR., TORIBARA, T. Y., AND WARNER, H., Anal.

Chem., 28, 1756 (1956). 23. KING, E. J., Biochem. J., 26, 292 (1932). 24. BURTON, K., Biochem. J., 62, 315 (1966). 25. SPIRO, R. G., AND SPIRO,M. J., J. Biol. Chem.,241,1271 (1966). 26. BROWN, A. H., Arch. Biochem., 11,269 (1946). 27. GOTTSCHALK, A., Chemistry and biology of sialic acids and

related substances, Cambridge University Press, New York, 1960, p. 33.

28. PIEZ, K. A., AND GROSS, I., J. Biol. Chem., 236, 995 (1960).

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Composition of Lens Capsule Vol. 244, No. 8

29. HARRINQTON, W. F., AND VON HIPPEL, P. H., Advan. Protein 34. MUKERJEE, H., SRI RAM, J., AND PIERCE, G. B., JR., Amer. Chem., 16, 1 (1961). J. Path&., 46, 49 (1965).

30. EASTOE, J. E., in G. N. RAMACHANDRAN (Editor), Treatise on 35. DOHLMAN, C.-H., AND BALAZS, E. A., Arch. Biochem. Biophys., collagen, Vol. I, Academic Press, New York, 1967, p. 1. 67, 445 (1955).

31. SPIRO, R. G., .I. Biol. Chem., 242, 1923,4813 (1967). 36. DRAKE, M. P., DAVISON, P. F., BUMP, S., AND SCHMITT, F. O., 32. SPIRO, R. G., Proceedings of tL Seventh International Congress Biochemistry, 6, 301 (1966).

of Biochemistry, Tokyo, 1067, Vol. III, p. 569. 37. KANQ, A. H., BORNSTEIN, P., AND PIEZ, K. A., Biochemistry, 33. SPIRO, R. G., AND FUKUSHI, S., J. BioZ. Chem., 244,2049 (1969). 6, 788 (1967).

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Suguru Fukushi and Robert G. SpiroThe Lens Capsule: SUGAR AND AMINO ACID COMPOSITION

1969, 244:2041-2048.J. Biol. Chem. 

  http://www.jbc.org/content/244/8/2041Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/244/8/2041.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from