British Joumal of Dermatology (1976) 94, 443.
Lysosomal hydrolases ofthe epidermis 4. OVERALL PROFILE IN COMPARISON WITH DERMIS AND OTHER TISSUES P.D.MIER AND JOSfi J.M.A.VAN DEN HURK Department of Dermatology, University of Nijmegen, The Netherlands Accepted for publication 30 June 1975
SUMMARY
The activities of fifteen acid hydrolases have been measured in seven tissues of the guinea-pig; fourteen of ±ese were also assayed in the epidermis of four other mammalian species. The most striking finding was that the proportion of acid phosphatase was consistently much higher in epidermis than in the other tissues investigated.
The function of lysosomes is, in a general sense, concerned with the degradation of biological macromolecules. Nevertheless, since there are wide differences in the turnover of macromolecules by the different tissues, one might expect to find corresponding variations in their patterns of hydrolase activity. Values for the acid hydrolase activities of most tissues, including epidermis, are now available. Unfortunately comparison of these figures is exceedingly hazardous, since different investigators have used different extraction techniques, selected different substrates and other reaction conditions, and have expressed the activities in terms of various reference variables (per unit fresh weight, dry weight, soluble protein, total protein, etc.). In addition significant species variation has been reported. We have therefore employed the fiuorimetric assay techniques described previously (Mier & v.d.Hurk, i975a-c) to obtain a direct comparison between the acid hydrolase profiles of epidermis and those of other tissues in the guinea-pig; we have also extended our study of epidermis to include a representative selection of mammalian species. Our findings are reported below. MATERIALS AND METHODS
Albino guinea-pigs (6-8 weeks) were killed by a blow to the head. Samples of tissue (Table i) were quickly removed and stored in liquid nitrogen prior to analysis. Epidermis was collected from all animals (Table 2) and from human subjects using a keratotome set for a depth of 0-2 mm. Homogenization was carried out in a solution of bovine serum albumin (i mg/ml) using a 'Micro-wet grinder' (Townson and Mercer) fitted with an ice-jacket. The homogenates were centrifuged to yield clear supernatants and dilutions prepared with bovine serum albumin solution. 443
444
P.D.Mier and J.J.M.A. van den Hurk TABLE I. Mean activities of fifteen acid hydrolases in each of seven tissues of the guinea-pig
EC no.
Enzyme
Liver
Kidney
Spleen
(2)
(2)
(I)
1400 53
1800 94
Acid phosphatase Pyrophosphatase Arylsulphatase A Arylsulphatase B a-glucosidase ;3-glucosidase a-galactosidase ^-galactosidase a-mannosidase jS-acetylglucosaminidase ^-glucuronidase Cathepsin Bi 3.4.4.3-4.4.9 Cathepsin C 3-4.4.23 Cathepsin D Arylamidase 3.5.I.-
3.1.3.2 3.1.4.1 3.I.6.1 3.1.6.2 3.2.1.20 3.2.1.21 3.2.1.22 3.2.1.23 3.2.1.24 3.2.1.30 3.2.1.31
6-1
3-0 6-2
II
3600 170
5-0 7-6
Intestine (2) , 840 24
4-7 75 85 44 77
170
360
17
51 270
410
540 99 6200 2400 450 8900 8-9 2900
240
no 260
5000 2900 350 4900 10
2800
84 2700 350 210
960 5-8 15000
99 52 2200
19 810
700 820
83 35
2000 49 2700
72 2-8
I IOO
97 0.81
5.7
6-2
240
IOO
310 I-I
360
(7)
(2)
(2)
2-2
260 400 170
Dermis Epidermis
Brain
56
0-057
0-46
o-it
13
2-1
53 35 7-8 38 7-8 25 8-1
1-2
1-6 18 23
36 8-7 34 58
22
42
0-68
2000
0-86 120
27
All values are in nmol/min/g wet wt tissue. Figures in parentheses indicate the numbers of different animals investigated; in the case of epidermis, this includes four values published previously (Mier & v.d.Hurk, I975a-c).
TABLE 2. Mean activities of fourteen acid hydrolases in the epidermis of various mammalian species EC no.
Enzyme
Acid phosphatase Arylsulphatase A Arylsulphatase B a-glucosidase )?-glucosidase a-galactosidase yS-galactosidase a-mannosidase ^-acetylglucosaminidase ^-glucuronidase Cathepsin Bi 3-4.4.Cathepsin C 3.4-4-9 3-4-4-23 Cathepsin D Arylamidase 3.5.I.-
3.1.3.2 3.I.6.1 3.1.6.I 3.2.1.20 3.2.1.21 3.2.1.22 3-2.1.23 3.2.1.24 3-2.1.30 3.2.1.31
Guinea-pig
Monkey
Cow
Rabbit
(7)
(2)
(2)
(2)
I IOO
140
5700
0-46
31
0-21
13 53
1-2
I-o
35 7-8 38 7-8 25 8-1 22
42
0-86 120
5-7 0-61 4-1 27
6-9 46 28 2-7 17 2-6 160
500
0-06 0-07
10
2-9
49 5-6 15 93
33
300 17 13 26
34 520
7-1
63 6-6 29 II
52 II
2-0
140
Human (27) 830
0-44 0-40 10
13
98 37 29 130 41
58 62 1-7 280
Values are in nmol/min/g wet wt tissue. Enzyme activities were measured using duplicate o-i ml samples of the tissue extracts. Except for the concentrations of the extracts (the optimum values of which were determined by preliminary experiments), the assay methods were in all respects exactly as described previously (Mier & v.d. Hurk, I975a-c).
Lysosomal hydrolases of the epidermis
445
RESULTS
The activides of fifteen hydrolases in seven different dssues of the guinea-pig are shown in Table i, and the acdvides of fourteen hydrolases from the epidermis of five mammalian species are compared in Table 2. DISCUSSION
Despite the enormous range of acdvides (more than 5 orders of magnitude) seen in Table i, certain patterns emerge. First, the 'profiles' of hydrolase acdvity of most dssues, although by no means idendcal, tend to be reladvely constant. Secondly, the absolute values are generally much higher for those dssues whose function involves the degradadon of large quanddes of exogenous macromolecules (liver, kidney, etc.) than those dssues which do not serve such a role (brain, connecdve dssue, epidermis). Neither of these findings is especially remarkable, being implicit in the lysosome concept as outlined by De Duve & Wattiaux (1966). Pardcular importance must therefore be attached to the one excepdon to this generalizadon, namely the extremely high level of phosphate esterase acdvity found in epidermis. This is better illustrated in Table 3, where the contribudon of each group of enzymes has been calculated as a percentage of the TABLE 3. Percentage contribution of each group of enzymes to the total hydrolase activity for the various tissues Enzyme group
Liver
Kidney
Acid phosphatase Pyrophosphatase Sulphatases Glycoside hydrolases Peptide hydrolases
94 049
024
005
64
o-o8
48
17
42
76
Spleen
Intestine
Brain
80 023 008
016 016
14
0-66 0-05 38 48
39 53
8-6
31
60
Dermis 34
0 29 006 24 42
Epidermis 76 39 012
6-6 13
total hydrolase acdvity. Expressed in this way, both the phosphate mono- and diesterases are an order of magnitude higher in epidermis than in any other dssue except dermis. It is possible that this last figure reflects contaminadon with a small percentage of keradnocytes originating from the hair follicles. Table 2 confirms earlier reports (e.g. Jansen & Hopsu-Havu, 1969) indicadng that real differences exist between the acid hydrolase distribudons in the skin of different species. Nevertheless, the high propordon of acid phosphatase remains a constant and general feature, the proportion of this enzyme varying between 31% (monkey) and 85% (cow snout). It is difficult to avoid the conclusion that the phosphate esterases of epidermis serve some function unique to this tissue; further evidence regarding this point is presented in the following paper (Mier et al., 1975). REFERENCES DE DUVE, C . & WATTIAUX, R. (1966) Functions of lysosomes. Annual Reviews of Physiology, 28, 435. JANSEN, C.T. & HOPSU-HAVU, V.K. (1969) Proteolytic enzymes in the skin. II. A comparative study of skin homogenates of five mammalian species. Acta dermato-venereologica, 49, 468. AiiER, P.D. & VAN DEN HuRK, J.J.M.A. (i975a) Lysosomal hydrolases of the epidermis, i. Glycosidases. British Journal of Dermatology, 9-3t ^-
446
P.D.Mier and J.J.M. A. van den Hurk
MIER, P.D. & VAN DEN HURK, J.J.M.A. (1975b) Lysosomal hydrolases of the epidermis. 2. Ester hydrolases. British Journal of Dermatology, 93, 391. MIER, P.D. & VAN DEN HURK, J.J.M.A. (1975c) Lysosomal hydrolases of the epidermis. 3. Peptide hydrolases. British Journal of Dermatology, 93, 509. MIER, P.D., COTTON, D.W.K., VAN DEN HURK, J.J.M.A. & JONCKHEER-VENNESTE, M . M . H . (1975) Lysosomal
hydrolases of the epidermis. 5. Variation with depth in the cow snout. British Journal of Dermatology, in press.
Lysosomal hydrolases ofthe epidermis 4. OVERALL PROFILE IN COMPARISON WITH DERMIS AND OTHER TISSUES P.D.MIER AND JOSfi J.M.A.VAN DEN HURK Department of Dermatology, University of Nijmegen, The Netherlands Accepted for publication 30 June 1975
SUMMARY
The activities of fifteen acid hydrolases have been measured in seven tissues of the guinea-pig; fourteen of ±ese were also assayed in the epidermis of four other mammalian species. The most striking finding was that the proportion of acid phosphatase was consistently much higher in epidermis than in the other tissues investigated.
The function of lysosomes is, in a general sense, concerned with the degradation of biological macromolecules. Nevertheless, since there are wide differences in the turnover of macromolecules by the different tissues, one might expect to find corresponding variations in their patterns of hydrolase activity. Values for the acid hydrolase activities of most tissues, including epidermis, are now available. Unfortunately comparison of these figures is exceedingly hazardous, since different investigators have used different extraction techniques, selected different substrates and other reaction conditions, and have expressed the activities in terms of various reference variables (per unit fresh weight, dry weight, soluble protein, total protein, etc.). In addition significant species variation has been reported. We have therefore employed the fiuorimetric assay techniques described previously (Mier & v.d.Hurk, i975a-c) to obtain a direct comparison between the acid hydrolase profiles of epidermis and those of other tissues in the guinea-pig; we have also extended our study of epidermis to include a representative selection of mammalian species. Our findings are reported below. MATERIALS AND METHODS
Albino guinea-pigs (6-8 weeks) were killed by a blow to the head. Samples of tissue (Table i) were quickly removed and stored in liquid nitrogen prior to analysis. Epidermis was collected from all animals (Table 2) and from human subjects using a keratotome set for a depth of 0-2 mm. Homogenization was carried out in a solution of bovine serum albumin (i mg/ml) using a 'Micro-wet grinder' (Townson and Mercer) fitted with an ice-jacket. The homogenates were centrifuged to yield clear supernatants and dilutions prepared with bovine serum albumin solution. 443
444
P.D.Mier and J.J.M.A. van den Hurk TABLE I. Mean activities of fifteen acid hydrolases in each of seven tissues of the guinea-pig
EC no.
Enzyme
Liver
Kidney
Spleen
(2)
(2)
(I)
1400 53
1800 94
Acid phosphatase Pyrophosphatase Arylsulphatase A Arylsulphatase B a-glucosidase ;3-glucosidase a-galactosidase ^-galactosidase a-mannosidase jS-acetylglucosaminidase ^-glucuronidase Cathepsin Bi 3.4.4.3-4.4.9 Cathepsin C 3-4.4.23 Cathepsin D Arylamidase 3.5.I.-
3.1.3.2 3.1.4.1 3.I.6.1 3.1.6.2 3.2.1.20 3.2.1.21 3.2.1.22 3.2.1.23 3.2.1.24 3.2.1.30 3.2.1.31
6-1
3-0 6-2
II
3600 170
5-0 7-6
Intestine (2) , 840 24
4-7 75 85 44 77
170
360
17
51 270
410
540 99 6200 2400 450 8900 8-9 2900
240
no 260
5000 2900 350 4900 10
2800
84 2700 350 210
960 5-8 15000
99 52 2200
19 810
700 820
83 35
2000 49 2700
72 2-8
I IOO
97 0.81
5.7
6-2
240
IOO
310 I-I
360
(7)
(2)
(2)
2-2
260 400 170
Dermis Epidermis
Brain
56
0-057
0-46
o-it
13
2-1
53 35 7-8 38 7-8 25 8-1
1-2
1-6 18 23
36 8-7 34 58
22
42
0-68
2000
0-86 120
27
All values are in nmol/min/g wet wt tissue. Figures in parentheses indicate the numbers of different animals investigated; in the case of epidermis, this includes four values published previously (Mier & v.d.Hurk, I975a-c).
TABLE 2. Mean activities of fourteen acid hydrolases in the epidermis of various mammalian species EC no.
Enzyme
Acid phosphatase Arylsulphatase A Arylsulphatase B a-glucosidase )?-glucosidase a-galactosidase yS-galactosidase a-mannosidase ^-acetylglucosaminidase ^-glucuronidase Cathepsin Bi 3-4.4.Cathepsin C 3.4-4-9 3-4-4-23 Cathepsin D Arylamidase 3.5.I.-
3.1.3.2 3.I.6.1 3.1.6.I 3.2.1.20 3.2.1.21 3.2.1.22 3-2.1.23 3.2.1.24 3-2.1.30 3.2.1.31
Guinea-pig
Monkey
Cow
Rabbit
(7)
(2)
(2)
(2)
I IOO
140
5700
0-46
31
0-21
13 53
1-2
I-o
35 7-8 38 7-8 25 8-1 22
42
0-86 120
5-7 0-61 4-1 27
6-9 46 28 2-7 17 2-6 160
500
0-06 0-07
10
2-9
49 5-6 15 93
33
300 17 13 26
34 520
7-1
63 6-6 29 II
52 II
2-0
140
Human (27) 830
0-44 0-40 10
13
98 37 29 130 41
58 62 1-7 280
Values are in nmol/min/g wet wt tissue. Enzyme activities were measured using duplicate o-i ml samples of the tissue extracts. Except for the concentrations of the extracts (the optimum values of which were determined by preliminary experiments), the assay methods were in all respects exactly as described previously (Mier & v.d. Hurk, I975a-c).
Lysosomal hydrolases of the epidermis
445
RESULTS
The activides of fifteen hydrolases in seven different dssues of the guinea-pig are shown in Table i, and the acdvides of fourteen hydrolases from the epidermis of five mammalian species are compared in Table 2. DISCUSSION
Despite the enormous range of acdvides (more than 5 orders of magnitude) seen in Table i, certain patterns emerge. First, the 'profiles' of hydrolase acdvity of most dssues, although by no means idendcal, tend to be reladvely constant. Secondly, the absolute values are generally much higher for those dssues whose function involves the degradadon of large quanddes of exogenous macromolecules (liver, kidney, etc.) than those dssues which do not serve such a role (brain, connecdve dssue, epidermis). Neither of these findings is especially remarkable, being implicit in the lysosome concept as outlined by De Duve & Wattiaux (1966). Pardcular importance must therefore be attached to the one excepdon to this generalizadon, namely the extremely high level of phosphate esterase acdvity found in epidermis. This is better illustrated in Table 3, where the contribudon of each group of enzymes has been calculated as a percentage of the TABLE 3. Percentage contribution of each group of enzymes to the total hydrolase activity for the various tissues Enzyme group
Liver
Kidney
Acid phosphatase Pyrophosphatase Sulphatases Glycoside hydrolases Peptide hydrolases
94 049
024
005
64
o-o8
48
17
42
76
Spleen
Intestine
Brain
80 023 008
016 016
14
0-66 0-05 38 48
39 53
8-6
31
60
Dermis 34
0 29 006 24 42
Epidermis 76 39 012
6-6 13
total hydrolase acdvity. Expressed in this way, both the phosphate mono- and diesterases are an order of magnitude higher in epidermis than in any other dssue except dermis. It is possible that this last figure reflects contaminadon with a small percentage of keradnocytes originating from the hair follicles. Table 2 confirms earlier reports (e.g. Jansen & Hopsu-Havu, 1969) indicadng that real differences exist between the acid hydrolase distribudons in the skin of different species. Nevertheless, the high propordon of acid phosphatase remains a constant and general feature, the proportion of this enzyme varying between 31% (monkey) and 85% (cow snout). It is difficult to avoid the conclusion that the phosphate esterases of epidermis serve some function unique to this tissue; further evidence regarding this point is presented in the following paper (Mier et al., 1975). REFERENCES DE DUVE, C . & WATTIAUX, R. (1966) Functions of lysosomes. Annual Reviews of Physiology, 28, 435. JANSEN, C.T. & HOPSU-HAVU, V.K. (1969) Proteolytic enzymes in the skin. II. A comparative study of skin homogenates of five mammalian species. Acta dermato-venereologica, 49, 468. AiiER, P.D. & VAN DEN HuRK, J.J.M.A. (i975a) Lysosomal hydrolases of the epidermis, i. Glycosidases. British Journal of Dermatology, 9-3t ^-
446
P.D.Mier and J.J.M. A. van den Hurk
MIER, P.D. & VAN DEN HURK, J.J.M.A. (1975b) Lysosomal hydrolases of the epidermis. 2. Ester hydrolases. British Journal of Dermatology, 93, 391. MIER, P.D. & VAN DEN HURK, J.J.M.A. (1975c) Lysosomal hydrolases of the epidermis. 3. Peptide hydrolases. British Journal of Dermatology, 93, 509. MIER, P.D., COTTON, D.W.K., VAN DEN HURK, J.J.M.A. & JONCKHEER-VENNESTE, M . M . H . (1975) Lysosomal
hydrolases of the epidermis. 5. Variation with depth in the cow snout. British Journal of Dermatology, in press.
