Histidine-rich protein of the keratohyalin granules

110

Biochimica et Biopl~vstca Acta, 719 (1982) 110-117 Elsevier Biomedical Press

BBA 21274

H I S T I D I N E - R I C H P R O T E I N OF T H E K E R A T O H Y A L I N G R A N U L E S S O U R C E OF T H E FREE A M I N O ACIDS, U R O C A N I C ACID AND P Y R R O L I D O N E CARBOXYLIC ACID IN T H E S T R A T U M C O R N E U M IAN R. SCOTT, CLIVE R. H A R D I N G and J O H N G. B A R R E T T

Unilever Research, Environmental Safety, Colworth laborato(v, Colworth House, Sharnbrook, Bedford MK44 1LQ ( U. K.) (Received January, 14th, 1982)

Key words: Histidine-rich protein; Keratohyalin granule," Amino acid," Urocanic acid; l~vrrolidone carboxvlic acid; (Stratum corneum)

The pool of free amino acids, urocanic acid and pyrrolidone carboxylic acid in mammalian stratum corneum has been shown to be derived principally or totally from the histidine-rich protein of the keratohyalin granules. The time course of appearance of free amino acids and breakdown of the histidine-rich protein are similar, as are the analyses of the free amino acids and the histidine-rich protein. Quantitative studies show that between 70 and 100% of the total stratum corneum-free amino acids are derived from the histidine-rich protein.

Introduction The normal mammalian stratum corneum contains a high concentration of water soluble, low molecular weight compounds which are principally amino acids or their derivatives such as urea, urocanic acid and pyrrolidone carboxylic acid [ 1,2]. These compounds have a vital role in preserving the integrity of the stratum corneum, particularly in maintaining its water content, and hence flexibility, in conditions of low humidity [3] and in absorbing harmful ultraviolet light [4]. Although it is known that environmental factors [5] and disease states [6] can influence the concentration of these compounds in the stratum corneum, it is not known what their source is, or what controls the level to which they accumulate. Early ideas that they were concentrated by evaporation of sweat have been disproved [7] and it is now generally accepted that they are either accumulated by epidermal cells prior to their cornification or that they arise from the breakdown of non-keratin proteins during the cornification process. 0304-4165/82/0000-0000/$02.75 ~) 1982 Elsevier Biomedical Press

Earlier work from this laboratory showed that free histidine and its derivative urocanic acid were not accumulated directly by the epidermal cells but were derived from the breakdown, within the stratum corneum, of proteins synthesized by the living cells of the epidermis [8]. We have since shown that pyrrolidone carboxylic acid, which accounts for approximately one third of all the amino acids or derivatives in the stratum corneum, is accumulated by a similar process [9]. It has been known for some time that the synthesis of the histidine-rich protein of the mammalian keratohyalin granule accounts for up to 40% of the total incorporation of histidine into epidermal protein [10]. We have shown that this protein is broken down to smaller proteins and peptides when the cells containing it enter the stratum corneum. This breakdown continues to very small peptides or free amino acids with a time course very similar to that of the formation of free histidine, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum [8,11]. We have therefore set out to test the hypothesis that this histi-

111 dine-rich protein is the sole, or major, source of all the amino acids and derivatives present in the stratum corneum. Methods

Analysis of stratum corneum-free amino acids. Dorsal skin from male albino guinea-pigs (Dunkin-Hartley, Colworth strain) was clipped and depilated with 'Zip Wax'. Layers of stratum corneum were obtained by stripping 2 × 1 inch areas using 'Ofrex Miracle Tape'. Up to three tape strips were obtained from each site. The strips were extracted in 5.0 ml of 2.0 M Aristar-HCl at 4°C for 3 days then the extract was filtered through a 0.45 /~ millipore filter and stored frozen. The urocanic acid content was determined as described previously [8] and amino acid analysis carried out using the lithium programme of a LKB 4400 analyzer. Pyrrolidone carboxylic acid contents were determined by analyzing duplicate samples after heating the extract at 100°C for 2 h. This procedure resulted in the increase of only the glutamic acid content. Assay of aspartate-4-decarboxylase. Epidermis and pure stratum corneum were isolated from guinea-pig dorsal skin using 'Dispase' as described previously [8]. The tissues were washed thoroughly in phosphate-buffered saline and homogenized in the proportion of 10 cmZ/ml of 0.1 M acetic acid/sodium acetate pH 5.0 containing 0.25 mM sodium pyruvate. Aspartate-4-decarboxylase activity was determined by measuring the release of |4C02 from [4-14C]aspartic acid (Amersham, U.K.) in an assay containing 60 ~1 of homogenate and 20 /xl of [4-14C] aspartic acid (1 mM, 50 /xCi/ml) essentially as described by Beaven, Wilcox and Terpstra [ 12].

Time course of histidine-rich protein breakdown and formation of histidine and urocanic acid. Guinea-pigs (400g, male) and albino rats (100g, male) were injected intradermally with [3H]histidine (40 /~1, 1 mCi/ml), killed after varying periods and the epidermis in a 1.7 cm diameter disc at the injection point was isolated by freeze-scraping [11]. Samples of epidermis were homogenized in 0.4 M perchloric acid (0°C, 30 min) and centrifuged (1500 × g, 4°C, 5 min). The soluble radioactivity was determined by counting aliquots of the

supernatant in a scintillator containing 10% soluene 350 (Packard), 1% water, 0.5% PPO and 0.02% POPOP in toluene. Thin layer chromatography of the perchloric acid extract showed that more than 90% of the radioactivity present was due to histidine and urocanic acid. The perchloric acid-insoluble protein was dissolved in soluene 350 and counted in the same scintillator. Duplicate samples of epidermis were homogenized in SDS lysis buffer (2% SDS, 0.1 M Tris-HC1 pH 8.1, 2 mM EDTA, 2 mM phenylmethylsulphonylfluoride and 25 mM dithioerythritol), heated at 60°C for 30 min, centrifuged ( 5 0 0 0 0 × g , 5 min, 15°C) and the solubilized radioactive proteins analyzed by SDSpolyacrylamide gel electrophoresis and fluorography as described previously [11].

Quantitative radioactive-labelling stud)' of the breakdown of the histidine-rich protein to free amino acids. Guinea-pigs (400 g, male) were clipped 3 days before the experiment commenced. They were then injected intradermally at four locations on the dorso-lateral skin with 40 ~1 of a mixture containing 42 /~Ci/ml L-[U-14C]histidine (339 m C i / m m o l ) and 177 /~Ci/ml L-[G-3H]glutamine (30 Ci/mmol). Groups of animals were killed at 5 and 24 h after injection, shaved and the epidermis isolated from 1.7 cm diameter biopsies and extracted in SDS lysis buffer as described above. The solubilized proteins were analyzed on 3.5% T, 1.3% C-15% T, 4.7% C gradient polyacrylamide gels, stained, and the band corresponding to the high molecular weight histidine-rich protein (band a, Fig. 1) cut out and counted in the above scintillator after extraction overnight at 60°C. The remaining animals were killed at 14 days after injection and the epidermis isolated and extracted in 0.4 M perchloric acid as described above. Aliquots of the supernatant were dried at 90°C prior to counting in the above scintillator. Analysis of the dried residue by TLC showed that more than 90% of the 3H activity was present as free glutamic acid, glutamine or pyrrolidone carboxylic acid and more than 90% of the 14C activity was due to histidine and urocanic acid. In addition to the epidermal samples taken from the injection sites, samples were taken from non-injected sites at least 2 cm away from any injection site. These were processed in the same way as the experimental samples to determine the

112

contribution of systemically absorbed radioactivity to the total epidermal radioactivity. In the case of the 5 h and 24h samples the contribution was negligible, for the 14-day samples it varied from 5-10%. This contribution was subtracted from the total radioactivity in the samples. Radioactivity was measured using a Packard PRIAS liquid scintillation spectrometer and dpm for both 3H and ~4C calculated using an external standard dual label channels ratio method optimized for the scintillator used. Quantitation of the amount of stratum corneum basic protein in superficial and deep rat stratum corneum. 100 g male albino rats were clipped and depilated with a single application of 'Zip Wax'. New born rats were taken within 12h of birth, when no depilation is needed. In both cases a layer of superficial stratum corneum was removed by a single stripping with 'Ofrex Miracle Tape' and the residual stratum corneum isolated using the enzyme 'Dispase' [8]. Both stripped and residual stratum corneum were extracted in 2% SDS, 0.1 M Tris-HC1 pH 8, 25 mM dithioerythritol, 2 mM EDTA and 2 mM phenylmethylsulphonylfluoride at 60°C for 30 min. The extracted proteins were analyzed on 3.5-15% gradient polyacrylamide gels [11] which were stained with PAGE Blue 83 (British

Drug Houses) and scanned at 594 nm using the densitometer attachment of a Pye Unicam SP8100 spectrophotometer. The amounts of the two major stratum corneum keratin polypeptides and of the stratum corneum basic protein were estimated by integration of the area under the respective peaks. Results

Time course of formation of histidine and urocanic acid and breakdown of the histidine-rich protein in the guinea-pig The interconversions of proteins, free histidine and urocanic acid derived from a single intradermal injection of [3H]histidine are shown in Fig. 1. For a period of 2 days, essentially all the radioactivity is present as protein, but during this period the pattern of radioactive proteins changes (Fig. l(a)). Initially it is dominated by the M r 340000 histidine-rich protein (a) which contains approx. 30% of the total protein radioactivity [11], the remaining activity being spread out over the gel in the keratins and minor proteins. Over the following few days the histidine-rich protein (a) breaks down progressively to the smaller proteins histidine-rich protein (b) and histidine-rich protein (c)) and hence to small peptides running at the dye

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Fig. 1. Interconversion of radioactive protein, histidine and urocanic acid after intradermal injection of [3H]histidine into guinea-pigs. Fig. la shows a stained gel and fluorogram of a 7.0% T, 1.3% C polyacrylamide gel on which were analyzed samples of proteins extracted with SDS lysis buffer from guinea pig epidermis pulsed with [3H]histidine between 2 h and 6 days earlier. Each track had the same total (protein+free amino acids) radioactivity applied. Fig. lb shows an analysis of similar epidermal samples with the proportion of the total epidermal radioactivity accounted for by proteins and free amino acids. IZI, Free histidine and urocanic acid; A , protein; K, major keratin polypeptide.

113 front on the gel. This breakdown has previously been shown to begin as the epidermal cells enter the stratum corneum [11]. After 3 days, free histidine and urocanic acid begin to be produced (Fig. lb) and at the same time the band of radioactive peptides in the get becomes diffuse, indicating that the peptides are approaching in size the point where they are no longer retained within the polyacrylamide gel. Between 3 and 6 days the level of histidine and urocanic acid increases to a stable maximum and the band of radioactive peptides disappears almost completely. Qualitatively, these results suggest a precursor/product relationship for the histidine-rich proteins and the free histidine and urocanic acid in the stratum corneum.

Amino acid analysis of the histidine-rich protein and the stratum corneum-free amino acids If the histidine-rich protein is the only, or the major, source of the stratum corneum free amino acids then there should be a good correlation between their amino acid analyses. Fig. 2a shows the comparative analyses for guinea-pig histidinerich protein (a) [11] and stratum corneum amino acids. There is an excellent correlation providing the following operations are performed on the data for the free amino acids, as shown in Fig. 2b.

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2. Comparison of the amino acid analyses of the histidinerich protein (a) and stratum corneum free amino acids in the guinea-pig. On the left are the actual analyses, showing the non-protein amino acids and derivatives found in the stratum corneum. On the right, those unusual compounds are added to the total for the amino acid from which they are derived. Fig.

(i) Combine the values for histidine and urocanic acid. This is valid as histidine is converted to urocanic acid in the stratum corneum by the enzyme histidase [8]. (ii) Combine the values for glutamine, glutamic acid and pyrrolidone carboxylic acid, all of which are glutamic acid derivatives. (iii) Combine the values for ornithine and citrulline with arginine. The enzymes catalyzing these two conversions have both been identified in the stratum corneum [ 13,14]. (iv) Combine the values for aspartic acid and alanine. To our knowledge, the enzyme aspartate4-decarboxylase which converts aspartic acid into alanine has not previously been demonstrated in the epidermis. Assays carried out as described under Methods showed however that guinea-pig epidermis had activity of 0.63 n m o l / h per cm 2 (30°C, 0.25 mM aspartic acid, pH 5) and isolated stratum corneum had an activity of 0.15 n m o l / h per cm 2. Thus the transformation of aspartic acid into alanine would be expected to occur in the stratum corneum. This connection between reduced aspartic acid and increased alanine content has also been made for human stratum corneum [15] but in that case aspartate-4-decarboxylase could not be detected. This difference may be due to the greater sensitivity of the assay method used in the present work. The comparative amino acid analyses are therefore consistent with the histidine-rich protein being the sole source of free amino acids and their derivatives in the stratum corneum.

of the

Fig. 1 shows qualitatively the relationship between the histidine-rich proteins and stratum corneum-free amino acids. To obtain a quantitative estimate of the extent of the contribution of histidine-rich protein (a) to the free amino acids it is necessary to compare the actual number of dpm's present in histidine-rich protein (a) after injection of radioisotope with the number present in free amino acids when a steady state is reached. Values expressed as percentages of the total radioactivity as in Fig. 1 can be misleading as the total epidermal radioactivity may fall over the time scale used, as proteins in the living epidermal cells

114 TABLE 1 QUANTITATIVE STUDY ON THE PASSAGE OF RADIOACTIVITY FROM THE HISTIDINE-RICH PROTEIN (a) INTO STRATUM CORNEUM-FREE AMINO ACIDS AFTER INJECTION OF RADIOACTIVE HISTIDINE AND GLUTAMINE

dpm in HRP (a) 4 hours after injection dpm in HRP (a) 24 hours after injeclion dpm in amino acids 14 days after injection

Number of samples

[3H]Glutamine (dpm/1.7 cm biopsy)

32

13243 + 2994

5917 , 1336

2.24

4

4536 ' 756

3186,

560

1.42

32

18853 . 7150

10047 ~ 3757

1.88

are broken down and resorbed by the body. This experiment was performed as a double label study to follow simultaneously the fate of histidine and glutamine. Because of the large variations in the total radioactivity taken up by the epidermis from an injection, large numbers of samples had to be analysed to give reliable data. The results are shown in Table I. Radioactivity in histidine-rich protein (a) 4 h after injection of the radioisotope mixture accounted for 70% of the 3H activity ultimately found as stratum corneum-free amino acids 14 days after injection but only 60% of the ~4C activity. Thus the remaining 30% of the )H and 40% of the ~4C found in the stratum corneum amino acids must come from some source other than the histidine-rich protein synthesized between 0 and 4 h after injection. There are two possibilities. Firstly, further radioactive histidine-rich protein may be synthesised later than 4 h after injection of the isotope due to the incorporation of residual radioactive free amino acid into protein or due to the recycling of radioactive amino acids from short lived epidermal proteins. Secondly, other epidermal proteins may make a contribution to the stratum c o r n e u m free amino acid pool. The first possibility would be consistent with the hypothesis that the histidine-rich protein is the sole source of the free amino acids, and with the data in Fig. 2. If this is true it would be predicted that the histidine-rich protein synthesized later than 4 h after injection must be relatively richer in [14C]histidine than in [3H]glutamine compared to

[14ClHistidine (dpm/I.7 cm b i o p s y }

Ratio [3H]/[14C]

the protein synthesized earlier, as it must account for the outstanding 40% of the 14C activity but only the 30% outstanding 3H activity. When histidine-rich protein (a) is isolated 24 h after injection, at which time most of the radioactive protein has broken down, and it is therefore enriched in the protein synthesised later than 4 h after injection, it does have the predicted relative excess of 14C over 3H (Table I). It can therefore be concluded that the breakd o w n of the histidine-rich protein (a) produces between 70 and 100% of the total stratum corneum free amino acids. Histidine-rich proteins in the rat

All the work referred to above relates to the epidermis of the guinea-pig. Much of the published data on histidine-rich proteins was obtained on the albino rat and the fate of these proteins in the rat differs in certain important respects from their fate in the guinea-pig. The initially synthesised, keratohyalin granule form referred to above as histidine-rich protein (a) is similar, although of slightly higher molecular weight [11] but on entering the stratum c o r n e u m it breaks down to produce a single low molecular weight protein referred to variously as H R P II [16] or stratum c o r n e u m basic protein [17,18]. This protein has been shown in vitro to cause the aggregation of keratin fibres into bundles [19] and has therefore been proposed to be the interfilamentous matrix protein of the stratum corneum. A role as a permanent structural protein would clearly be incom-

115

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Fig. 3. Interconversion of radioactive protein, histidine and urocanic acid after the intradermal injection of [3H]histidine into 100g male albino rats. The analyses are the same as shown for Fig. I. D, Free histidine and urocanic acid; A, protein; K, major keratin polypeptide; SCBP stratum corneum basic protein.

patible with the role suggested above as b e i n g a source of free a m i n o acids. Some of the experim e n t s described above were therefore repeated on the rat to test whether the histidine-rich protein had a similar fate in this species. Fig. 3 shows the i n t e r c o n v e r s i o n of the high molecular weight histidine-rich protein, the strat u m c o r n e u m basic protein ( H R P II) a n d free histidine a n d urocanic acid p r o d u c e d after the i n t r a d e r m a l injection of [3H]histidine into rat skin. The p a t t e r n of b r e a k d o w n is very similar to, although simpler than, that shown for the guinea-pig in Fig. 1. Similar results (not shown) were o b t a i n e d using new b o r n rats rather than the older animals. It is clear therefore that the histidine-rich proteins have a similar fate in the rat to that d e m o n s t r a t e d in the guinea-pig a n d that they do not constitute a p e r m a n e n t structural c o m p o n e n t of the s t r a t u m corneum.

Distribution o f stratum corneum basic protein between the superficial and deep stratum corneum o f the rat The results quoted above would indicate that the s t r a t u m c o r n e u m basic p r o t e i n should be f o u n d

only in the deeper parts of the rat s t r a t u m corneum. T o c o n f i r m this, superficial s t r a t u m c o r n e u m was t a k e n from b o t h m a t u r e a n d n e w - b o r n rats by tape stripping a n d the a m o u n t s of s t r a t u m c o r n e u m basic p r o t e i n a n d keratin in b o t h this superficial a n d the residual deeper s t r a t u m c o r n e u m measured as described in M e t h o d s (Table II). I n the

TABLE II DISTRIBUTION OF STRATUM CORNEUM BASIC PROTElN BETWEEN THE SUPERFICIAL AND LOWER STRATUM CORNEUM OF THE RAT SCBP, stratum corneum basic protein. % of total keratin

Ratio SCBP/ keratin

Newborn rat Superficial stratum corneum Lower stratum corneum

2.2 97.8

0.47 0.83

Adult rat Superficial stratum corneum Lower stratum corneum

11.7 88.3

0.08 0.83

116

mature rat there are only traces of the stratum corneum basic protein in the superficial stratum corneum as expected. However, in the newborn animal, the basic protein appears to be present at quite high levels in the most superficial stratum corneum. This apparently contradictory result is not, in fact, in conflict with the evidence shown in Fig. 3, that the basic protein is short lived. In the development of the rat foetus, the first layer of true stratum corneum only appears 1 day before birth [20]. As a result, at birth insufficient time has passed for the stratum corneum basic protein to have begun to break down in the newborn animal. The stratum corneum of the newborn rat is thus an immature structure in which the later stages of stratum corneum development have not had time to occur. This immaturity probably also accounts for the very small amount of the stratum corneum that is removable by tape stripping, because the desquamation process is at a very early stage. Discussion

Keratohyalin granules occupy a large part of the epidermal cell shortly before it undergoes cornification and they disappear as the cell enters the stratum corneum. Labelling studies indicate that synthesis of the histidine-rich proteins which are major components of these granules occurs at a greater rate even than the keratins and the only reason that the keratins predominate in the epidermis is the short lifetime of the histidine-rich proteins [11]. Despite their obvious quantitative importance however, functions for the keratohyalin granules have until recently not been established. The work described in this paper establishes one such function. The keratohyalin granules are the repository of an insoluble protein which is designed to break down rapidly and completely after the cell containing the protein has entered the stratum corneum. The significance of such an indirect method of generating the large free amino acid pool of the stratum corneum should not be underestimated. On entering the stratum corneum, the epidermal cell forms around itself a protein envelope covalently cross-linked by the action of transglutaminase [21] and within this envelope the keratin fibres aggregate and become cross-linked by disulphide bonds [22]. This produces a very

strong and elastic structure capable of maintaining the extremely flattened shape of the cell. The generation of a large pool of low molecular weight species in this cell would set up a very large osmotic pressure under the conditions of high water activity found deep in the stratum corneum [23]. In such a flattened structure, this pressure would lead to large tensional forces in the plane of the cell which would be transmitted from cell to cell by the inter-squame junctions present [24]. Thus the sheets of cells deep in the stratum corneum would be under a continuous tension which would only lessen as the cells moved into areas of lower water activity nearer to the skin surface. This tension would have a major significance to the elastic properties of the whole stratum corneum. Another function which has recently been proposed for the histidine-rich proteins of the keratohyalin granules is that of an inactive, phosphorylated, precursor of the stratum corneum basic protein which has been shown to stimulate the aggregation of keratin fibres in vitro [19,25]. As shown above, the stratum corneum basic protein has only a short lifetime after its formation from its high molecular weight, phosphorylated, precursor and cannot therefore be a permanent structural component of the stratum corneum. This is not, however, incompatible with the protein having a role in aggregating and ordering keratin fibres in the stratum corneum, since shortly after the epidermal cell enters the stratum corneum, the keratin fibres become extensively cross-linked by disulphide bonds. This would stabilize the new, close-packed, keratin structure and hence perhaps make the continued presence of the stratum corneum basic protein unnecessary. The existence of a discontinuity in the stratum corneum between a lower layer of cells in which the keratin fibres are surrounded by a keratohyalin derived matrix protein and upper layers where the matrix material is degraded is supported by morphological studies. Brody [26,27] showed that only in the lower part of the guinea-pig stratum corneum was the cell filled with the 'keratin pattern' of fibres in a dark stained matrix. In the upper layers this clear 'keratin pattern' becomes irregular or diffuse and large apparently empty spaces appear,

117

These findings are consistent with our findings that the stratum corneum histidine-rich proteins are short lived. However, Brody also showed that small amounts of 'keratin pattern' persisted in the upper stratum corneum. The completeness of the degradation of radioactive histidine-rich proteins shown in Figs. 1 and 3 make it unlikely that the matrix of this remaining 'keratin pattern' consists of unmodified histidine-rich protein. There are at least two possible explanations consistent with these data. Firstly the matrix material may be histidine-rich protein that has become covalently linked to the keratin fibres, perhaps by the action of transglutaminase. The amount of radioactivity in the SDS/dithioerythritol insoluble material in experiments such as that shown in Fig. 1 is however less than 10% of that found initially in the histidine-rich proteins [11]. Since much of this radioactivity must be accounted for by the proteins of the stratum corneum cell envelope, such covalently bound histidine-rich protein could only be a very minor component of the upper stratum corneum. The second possible explanation is that proteins other than the histidine-rich proteins contribute to the matrix material. This is not an unlikely possibility as there have been repeated observations of the involvement of sulphur-rich proteins in the final stages of epidermal differentiation [28,29]. In conclusion we have shown that the histidine-rich proteins are likely to have a dual role in the formation of the normal healthy stratum corneum. Initially they are probably involved in the organisation and aggregation of keratin fibres [19] but later they break down to generate in situ the large pool of amino acids and derivatives so important to maintaining the flexibility and integrity of this vital barrier.

References 1 Tabachnick J. (1959) J. Invest. Dermatol. 32, 563-568 2 Tabachnick J. and LaBodie, J.H. (1970) J. Invest. Dermatol. 54, 24 31 3 Middleton J.D. (1977) Cosmet. Toilet. 92, (5), 34-40 4 Anglin J.H. (1976) Cosmet. Toilet. 91, 47-49 5 Pratzel H. and Fries P. (1977) Arch. Dermatol. Res. 259, 157-160 6 Flesch P., Hodgson C. and Jackson Esoda, E.C. (1962) Arch. Dermatol. 85, 476-484 7 Dowling G.B. and Naylor P.F.D. (1960) Brit. J. Dermatol. 72, 57-61 8 Scott, I.R. (1981) Biochem. J. 194, 829-838 9 Barrett, J.G. and Scott, I.R. (1982) J. Invest. Dermatol., in the press 10 Bernstein I.A. (1970) J. Soc. Cosmet. Chem. 2 1 , 5 8 3 - 5 9 4 11 Scott I.R. and Harding C.R. (1981) Biochim. Biophys. Acta 669, 65 78 12 Beaven M.A., Wilcox G. and Terpstra G.K. (1978) Anal. Biochem. 84, 638-641 13 Pratzel H. and Geiger K. (1977) Arch. Dermatol. Res. 259, 151-156 14 Kubilus J. and Baden H.P. (1978) Fed. Proc. 37, 1780 15 Pratzel H., Chlebarov S. and Dana P. (1978) Arch. Dermatol. Res. 261, 95 16 Ball R.D., Walker G.K. and Bernstein I.A. (1978) J. Biol. Chem. 253, 5861-5868 17 Dale B.A. (1977) Biochim. Biophys. Acta 491, 193-204 18 Dale B.A., Vadlamudi B., Delap L.W. and Bernstein I.A. (1981) Biochim. Biophys. Acta. 668, 98-106 19 Dale, B.A., Holbrook K.A. and Steinert P.M. (1978) Nature 276, 729-731 20 Stern, I.B., Dayton, L. and Duecy, J. (1971) Anat. Rec. 170, 225 234 21 Rice R.H. and Green H. (1977) Cell 11,417-422 22 Sun T.T. and Green H. (1978) J. Biol. Chem. 253, 2053-2060 23 Stockdale M. (1978) J. Soc. Cosmet. Chem. 29, 625-639 24 Allen T.D. and Potten C.S. (1975) J. Uhrastruct. Res. 51, 94-105 25 Lonsdale-Eccles J.D., Haugen J.A. and Dale B.A. (1980) J. Biol. Chem. 255, 2235-2238 26 Brody, 1. (1959) J. Uhrastruct. Res. 2, 482-511 27 Brody, I. (1960)Acta Dermato-Venereol. 40, 74-84 28 Matoltsy, A.G. and Matohsy, M.N. (1972) J. Ultrastruct. Res. 41, 550-560 29 Jessen, H. (1973) Histochemie 33, 15 29