The Synthesis of Epidermal Proteins

THE SYNTHESIS OF EPIDERMAL PROTEINS* II. SOLUBLE PROTEINS

SIMON ROTHBERG, Pn.D.

Rudall (1) demonstrated that different epi- site in the epidermis or synthesis of the specific dermal proteins were present in longitudinal soluble proteins occurs throughout all levels of sections of cow snout epidermis. The hypothesis

the epidermis.

that different epidermal proteins are present at different levels of the epidermis was further investigated by us in a previous work (2). Epidermal proteins, labelled by the systemic administration of glycine-1-C'4, appeared at the

EXPERIMENTAL

Tissue Collection and Preparation Scales of psoriasis were collected from (the bed

linens of) three psoriatic patients daily for ten

skin surface at sequential times. Of several days following a single administration of 10-

microcuries of glycine 1C14. The patients were maintained during the study on a 2600 calorie, purine free, 70 gram protein diet. Therapy for the psoriasis consisted of topical applications of an ointment made of equal parts of Aquaphor® and

proteins isolated, a protein fraction precipitated at pH 4.5 was found to be the protein showing

earliest peak incorporation of the label. The hypothesis that distinct proteins are synthesized in distinct cell layers of the epidermis was made

water. During this period the use of soap and

in this report. A possible correlation of this

water on the skin was prohibited.

hypothesis with electron microscopic observations

of the procedures previously described (7). The

The tissue was processed with some modification

may be made. Electron microscopy findings dry psoriatie scales were first defatted in ether and (3, 4, 5, 6) indicate that events during keratini- ground in a Wiley mill to 60 mesh particle size. The

zation reflect continuous changes occurring in the powdered tissue, suspended in 0.1 M phosphate pH 7.3, was dialyzed at 00 against phoscells as they travel tbrough the epidermis. The buffer, phate buffer to remove amino acids, peptides and fibrillar structures appearing during these kera- other diffusible substances. Fresh phosphate buffer tinization events may possibly result from for dialysis was supplied after 3, 19, and 22 hours, aggragation of structurally complementary pro- after which the dialysis residue was centrifuged at teins synthesized at succeeding levels of the 3000 R.P.M. for 15 minutes. The supernatant solution plus two washes, constitute what will be reepidermis. ferred to as the soluble proteins of the sac superThe epidermal proteins previously dealt with natant. The dialysis residue, after the phosphate (2) were isolated by solubilization of the normally buffer was removed by dialysis, was processed as insoluble proteins and fractionating these proteins shown in Figure 1 in order to obtain the 'solu-

proteins'. The sac supernatant proteins into four different fractions. The epidermal bilized were separated by ammonium sulfate (AMS) proteins fractionated in the present work are fractionation and precipitates obtained at 20, 40,

those normally soluble in phosphate buffer, pH 50, 60 and 70% saturation. This is shown on the 7.3. The relationship between these two groups right side of Figure. 1

of epidermal proteins is at present unclear.

Isolation of Glycine from Hydrolyzed Protein Twenty milligrams of dry protein (when avail-

However, if the soluble proteins were to show a

pattern of synthesis similar to the insoluble proteins it would suggest that the soluble proteins

possibly may be precursor proteins to the insoluble proteins. Absence of any pattern of sequential synthesis would suggest either that the soluble proteins are synthesized at a single * From the Dermatology Branch, National Cancer Institute, Bethesda, Maryland. National Institutes of Health, Public Health Service, U. S. Department of Health, Education and Welfare. Technical Assistance given by John L. Lee, Lem Davis, Jr. and Gertrude D. Axilrod. Received for publication November 14, 1963.

able) were suspended in 20% HC1 (1 ml. per 10 mg.

protein) and hydrolyzed in a sealed ampoule at 1050 for 16 hours. The hydrochloric acid was removed in vacuo and the residue, after redissolving in water, was dried in vacuo. The hydrolysate was

taken up in a minimum volume of water and streaked on two sheets of Whatman 3, 22)4" by 16". Appropriate amino acid markers were also spotted on the papers to permit clear identification

of glycine. The papers were saturated with 3% (v/v) formic acid and run for one hour at 2500 volts, in a conducting solution of 3% formic acid, pH 2.6; the non-conducting coolant was Varsol®,

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Psoriasis Scale, defatted, 60 mesh. 500 mg. suspended in 20 ml. 0.1 H phosphate buffer, pH 7.3. Placed inside dialysis sac. Dialyzed

at 0°C. against 200 ml of buffer with magnetic stiring.

Dialy::::i:lDiaYsis Sac Contents (amino acids, peptides, etc.)

3000 RPM

SAC RESIDUE( Dialyses against H 0. Suspended in 40 ml. 0.05 H NaOH. (0.8 1. per 10 mg. orig weight) Shaken for 20 hours, at 380 under nitrogen gas.

Sac Supernatant (Fraction S.S.) Dialysed against H,0. To 207. saturation with fM* 3000

RPM

Residue A

Fraction A

S.S. 0%**

QxGG

Black residue

to 40% A14S Saturation

3000 H

Fraction A

Dialyse

S.S.-A

S.S.-B

S.S. 07.

against H20 to

pH 6.5.

to 507. AM Saturation 0.1 H HC1 to ph

Fraction

0.1

A **

5.5

A

to

enatant

H HCI to pH 5.0 S.S.-D

S.S. 607.

RPM

to 707,

2

0.1

607. AM Saturation

AAS Saturation

H HCI to pH 4.5

RPM__

FrTnA'3 (4.5)

S.S. 70%

S.S.-E

A3

i All centrifugation at 00. * All AMS addition at 00 and material

allowed to stand over night at 00 before

separation.

** All fractions redissolved in 0.1 M (NH4)2C03, and dialysed free of salt and lyophilized to

dryness. FIG. 1. Procedure for obtaining protein fractions from psoriatic scale

maintained at 35° to 40°. 7% formic acid used in the latter part of this work was equally as effective. The paper was dried in a chromatographic oven at 60° and separated marker strips developed in 0.4% (w/v) ninhydrin in acetone. The identified glycine sector was eluted with water and an aliquot rerun in another chromatographic system to verify that glycine was the only amino acid present. As can be seen from Figure 2, possible contaminants in this system are histidine or alanine. The solvent system, n-butanol-acetic acid-water

(4:1:5 by volume) or methanol-water-pyridine

(20:5:1 by volume) were used to indicate the ab-

sence of contaminant. When contaminant was present the glycine was re-run in one of these systems to complete the purification. Measurement of Glycine Activity

When the presence of only glycine was assured,

an aliquot of this amino acid was quantitated by the ninhydrin method of Moore and Stein (8). The

remainder, usually one milligram or less, was plated and counted on fiat aluminum disks or pans, 1w" in diameter (Nuclear Chicago Corpora-

SYNTHESIS OF EPIDEEMAL PROTEINS. II

161

FIG. 2. High voltage eleetrophoresis of epidermal protein hydrolysate (2500 volts, 1 hour, 35—40°, in 3% (v/v) formic acid; pH 2.6).

tion, Chicago, Illinois), as an infinitely ttin the three samples are graphically represented in

sample in an automatic gas flow low background Figures 3—5 wherein the glyeine specific activities counter (Model C-us Low Background Automatic sample changer, Model 186 Scaler and Model C- are plotted against time following administration

111B printing timer, all from Nuclear Chicago of the glycine 1-C". The glyeine isolated from Corporation, Chicago, Illinois).

these proteins had different peak activities for the different protein fractions.

RESULTS

From each of the three samples used in these experiments nine different proteins have been separated. Five of these proteins were obtained from the phosphate buffer, pH 7.3, as soluble proteins while the other four were obtained from the 'solubilized proteins'. In this work experi-

nIscUSSION

In the three patients studied the ammonium sulfate fraction precipitating at 50% saturation

shows peak activity at three days. The 40%

fraction, on the other hand, seems consistently to have the latest peak activity at four to five mental data on the soluble proteins was ob- days. The 60% and 70% fractions do not show a consistency in peak activity that can be corretained. The experimental data obtained for each of lated readily with a distinct synthesis level in the

162

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY SPECIFIC ACTIVITY OF GLYCINE ISOLATED FROM SOLUBLE PROTEIN FRACTION OBTAINED FROM PSORIATIC SCALE AFTER ADMINISTRATION

OF IOC GLYCINE—I-C'4

45

SPECI FIC

ACTIVITY cpm 1mg

glycine

I

2

3

4

5

6

7

8

9

10

DAYS AFTER ADMINISTRATION

FIG. 3. Plot of specific activity of glycine isolated from soluble protein fractions (obtained from psoriatic scale) after administration of lOgiC glycine 1-C'4. Patient M.

SPECIFIC ACTIVITY OF GLYCINE ISOLATED FROM SOLUBLE PROTEIN FRACTION OBTAINED FROM PSORIATIC SCALE AFTER ADMINISTRATION OF IOJLC GLYCINE- I-C'4

SPECIFIC

ACTIVITY cpm mig

glycine

FIG. 4. Plot of specific activity of glycine isolated from soluble protein fractions (obtained from psoriatic scale) after administration of 10m0 glycine 1-C'. Patient J. F.

163

SYNTHESIS OF EPIDERMAL PROTEINS. II SPECIFIC ACTIVITY OF GLYCINE ISOLATED FROM SOLUBLE PROTEIN FRACTION OBTAINED FROM PSORIATIC SCALE AFTER ADMINISTRATION

OF lOuC GLYCINE-I-C'4

4

SPECIFIC

ACTIVITY C pm/mg

glycine

I

2

3

4

5

6

7

8

9

10

DAYS AFTER ADMINISTRATION

Fiu. 5. Plot of specific activity of glycine isolated from soluble protein fractions (obtained from psoriatic scale) after administration of l0pC glycine 1-C'4. Patient B. P.

epidermis. In fact, the results may indicate that the latter fractions are synthesized throughout the viable epidermis. However, any correlation with protein synthesis at distinct levels of the epidermis must assume that the rates of synthesis

epidermis then similarities in day of peak activity

of these fractions to that of the solubilizcd fractions might suggest a relationship between

the two. The "50% fraction" of the soluble proteins has the shortest time for peak activity,

and the amino acid pools supplying the individual similar to that found for the solubilized "fraction 4.5". A further similarity between these two proteins are equal. The different peak activities for these fractions protein fractions is that each precipitates at pH

is similar to the results obtained with the solu- 4.5. These similarities, however, seem entirely bilized fractions of the insoluble proteins. The fortuitous, since their amino acid compositions site or sites of synthesis of the soluble proteins arc quite different, mainly in cystine, proline and in psoriatic epidermis is unknown. rphe possibility

glutamic acid contents (10).

A possible correlation of the conclusions of transported into the epidermis from the serum. this work, that synthesis of specific proteins The soluble proteins, however, if derived ex- occurs at specific levels of the epidermis, with clusively from serum proteins would be expected electron and light microscopic findings may be to have similar days of peak activity. In addition, made. Both of the latter tcchnics indicate that these proteins when tested by the Ouchterlony during keratinization there is continuous change technic (9), do not form prccipitin reactions with in the cells throughout the epidermis. At the basal cell layer the protein synthesized forms the horse antibuman whole serum'. Tithe soluble fractions are synthesized in the tonofilaments (11). As the tonofilaments aggre-

exists that some or all of these proteins are

gate to form tonofibrils, an interfilamcntous

'The author is indebted to Dr. Kenneth Blay- structure (12) becomes evident. This transition lock, Dermatology Branch, NCI for carrying out of tonofilaments to tonofibrils may occur sponthese experiments.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

taneously because of the chemical or physical nature of either the tonofilamentous protein, the

interfilamentous material, or both. It is also possible that these aggregations are catalyzed by specifically operative enzyme systems. Whether

the soluble proteins are the interfilamentous material is not known. sUMMARY

1. The soluble proteins of exfoliated epidermal psoriasis scales were fractionated with ammonium sulfate to give five major fractions.

2. These proteins, labelled by the administration of glycine 1-C' to the patient, appeared

at the skin surface at different times. In all three cases a fraction precipitated by 50% ammonium sulfate was the first to show peak incorporation of radioactivity, and a fraction precipitated at 40% saturation was the last to show peak incorporation.

3. The results indicate that in the epidermis, synthesis of distinct soluble proteins occurs at distinct epidermal levels, and is similar in this regard to the synthesis of insoluble proteins.

2. ROTHBERG, S.: Synthesis of epidermal proteins. I. Alkali Solubilized (insoluble) Proteins I Invest. Derm., 43: 151, 1964. 3. SELBY, C. C.: The fine structure of human epi-

dermis as revealed by the electron micro-

scope. J. Soc. Cosm. Chem., 7:584, 1956. 4. ODLAND, G. G.: The fine structure of the interrelationship of cells in the human epidermis. J. Biophys. Biochem. Cytol., 4: 529, 1958.

5. Bitorw, I.: An ultrastructural study on the role of the keratohyalin granules in the keratinization process. J. Ultrastruct. Res.,

4: 264, 1960. 6. ZELIcicsoN, A. S. AND HARTMANN, J. F.: An

electron microscopic study of human epi-

dermis. J. Invest. Derm. 36: 65, 1961. 7. RoTirssjaa, S., Caouaisi, R. G. AND LEE, J. L.:

GlycineCi4 incorporation into the proteins

of normal stratum corneum and the abnormal stratum corneum of psoriasis. J.

Invest. Derm, 37: 497, 1961. 8. Moona, S. AND STEIN, W. H.: A modified

ninhydrin reagent for the photometric determination of amino acids and related

compounds. J. Biol. Chem., 211: 907, 1954. 9. OUDIN, J.: Methods in Medicel Research, pp. 5, 335. Ed. by M. Cohn, Chicago, Yearbook Publishers, 1951. 10. ROTIrBEEG, S. AND CnouNsa, R. C.: Unpublished observations, 1963.

11. BRODY, I.: An ultrastructural study on the

role of the keratohyalin granules in the keratinization process. J. Ultrastruct. Res., 3: 84, 1959.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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