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S-100 protein in adipose tissue
Iur. J. Emhem. Vol. 15, No. 5. pp. 609-613. Printed in Great Britain. All rights reserved
0020-7 I I X;83/050609-05$03.00,0 Copyright 0 1983 Pergamon Press Ltd
1983
S-100 PROTEIN
IN ADIPOSE
TISSUE
K. KATCI’*, F. SUZUKI’ and T. NAKAJIMA*
I Department
of Biochemistry,
‘Pathology
Division,
Institute for Developmental Research, Aichi Prefectural Kasugai. Aichi 480-03, Japan and National Cancer Research Institute, Tokyo 104, Japan (Received
10 Auyust
1982)
Abstract-l. The nervous system-specific S-100 protein is present at high levels various animal species. 2. The adipose S-100 protein levels in beef and rat were 1.13 and 1.59 pg/mg which were comparable to the brain levels (beef, 4.85 pg/mg; rat. 1.98 pg/mg). 3. Adipose tissues of rabbit, pig and human contained 0 222, 0.149 and 0.083 respectively. 4. The S-100 protein level in rat adipose tissue showed a developmental profile brain.
INTRODUCTION
S-100 protein is a nervous system-specific protein, and it has been reported to be localized mainly in the cytoplasm of glial cells in nervous tissues (Moore, 1975). However, recent reports from several laboratories revealed immunocytochemically that the S-100 protein is also present in non-nervous tissues, such as interstitial cells of the pineal gland (Moller et al., 1978). satellite cells of the adrenal gland (Cocchia & Michetti. 1981), stellate cells of the hypophysis (Nakajima et ul., 1980), melanocytes and Langerhans cells of human skin (Cocchia ef al., 1981) and human chondrocytes (Stefansson et al., 1982). Recently, we have prepared a highly sensitive enzyme immunoassay system for S-100 protein (Kato et (I!., 1982). In researching the tissue distribution of S-100 protein with the immunoassay method, we found that rat adipose tissue and cartilage contained high levels of S-100 protein comparable to those in the central nervous tissue (Suzuki et al., 1982). In this report, we describe the levels of S-100 protein in adipose tissues from various animal species and the developmental profile of S-100 protein level in rat adipose tissue.
MATERIALS
AND
METHODS
Colony,
Emyme
immunoassay
in adipose protein,
tissues
of
respectively.
pg S-100 proteinimg, similar
to that in the
fbr S- IO0 protein
The enzyme immunoassay system for S-100 protein was composed of a silicone rubber solid-phase (413 x 4 mm) with immobilized (anti-beef S-100) antibodies and the antibodies labelled with p-D-galactosidase from Escherickia coli as described recently (Kato et al., 1982). Samples containing S-100 protein were incubated at 30°C for 5 hr with a piece of the solid-phase, and then the solid-phase with bound S-100 protein was reacted with the enzyme-labelled antibodies at 4°C overnight. From the fi-o-galactosidase activity bound on the solid-phase, amounts of S-100 protein could be determined with a minimum detectable sensitivity of 3 pg beef S-100 protein (Kato ct al., 1982). Standard
S- 100 protein
Immunologically, S-100 protein is a species-nonspecific protein, and the antiserum to beef S-100 protein crossreacts with S-100 proteins from various animal species (Moore, 1975). Therefore, we have determined the S-100 protein levels in brains and adipose tissues from various animals with the above immunoassay system by use of the purified beef S-100 protein as a standard. and the results are expressed as pg or ng of beef S-100 protein equiva-
lents/mg protein or g wet weight. Concentrations of the purified S-100 protein were estimated by using a value E:& = 3.44 (Calisson et al., 1969). Protein concentrations of the extract were determined with Bio-Rad Protein Assay (Bio-Rad), which utilizes a principle of protein-dye binding (Bradford, 1976). Tissue extract
S-100 protein was purified from beef brain by the method of Uyemura et al. (1971) and the final preparation was electrophoretically pure as described previously (Nakajima et al.. 1980). The antiserum against S-100 protein was raised in New Zealand white rabbits by injecting the purificd S-100 protein with methylated bovine serum albumin ~1sdescribed by Haglid ct al. (1977). The antiserum was found to be specific to S-100 protein showing a single precipitin line on double immunodiffusion against a crude extract of beef brain (Fig. 1).
*TO whom correspondence should be addressed: Dr Kanefusa Kate. Department of Biochemistry, Institute for Aichi Prefectural Colony. Developmental Research. Kamiya. Kasugai. Aichi 4X&03. Japan.
Fresh brains and abdominal adipose tissues of beef and pig were obtained from a slaughter house. Female rats (Wistar) aged 2-10 weeks, male adult mice (129/SvJ) and a male rabbit were sacrificed and whole brains and abdomlnal adipose tissues were quickly removed. Human tissues were obtained on the autopsy. All samples were kept frozen at -80°C until analysis. The frozen tissues were homogenized at 0°C in 4-vol. (v/w) of 0.05 M Tris-HCI buffer, pH 7.0, containing 0.1 M NaCl with a glass homogenizer. The homogenate was centrifuged at 4-C at 107,OOOg for 1 hr, and the supernatant was used for the assay of S-100 protein. Each extract was variously diluted (50,00&50-fold) with 0.01 M sodium phosphate buffer, pH 7.0 containing 0.3 M NaCI, 1 mM MgCl*, 1 mM calcium acetate, O.l”,, bovine serum albumin (Armour Phar-
610
K. KATO
maceuticais). OS”,, gelatin (Difco Laboratories) and O.l”,, NaN, at room temperature. Aiiquots (0.5 ml) of the diluted samples were subjected in duplicate to the immunoassay as described above.
Doubic ~mmunodiffusion of the crude extract against the antiserum to beef S-100 protein was performed at room temperature (l&)-25’C) on a slide piass (2.5 x 7 cm) covered w& 3 ml of l”,, agarose containing 0.9”, NaCI, I mM calcium acetate and 20mM Tris HCI buffer, aH 7.0. After variously concentrated (i&200-fold with M’inicon B-15. Amicon Corp.). 5 iti each of the crude extract were applied on the wells. Immunoeiectrophoresis was carried out on the slide glass covered with 3 ml of i.2”,, agarose in 60 mM barbital buffer. pH 8.6 at 4 C. Run time was 4t?min at 3 mA/slide glass. After eiectrophoresis. anti-S-100 serum was applied to the trough at room temperature. Precipitin lines were observed after 24 hr.
RESVLTS AUD
DISCUSSION
Lecels cf S-l 011 grofein in adipose animal species
tissues qf‘ cariaus
The immunoassay of S-100 protein in crude extracts was performed with the variously diluted samples at two or three different concentrations of each extract, and the results were expressed as means of the assays. As shown in Table 1, S-100 protein is present in adipose tissues of beef and rat at high levels comparable to those of central nervous tissues when compared on the basis of mg protein. Adipose tissues of rabbit, pig and human also contain considerably high levels of S-100 protein, since most of rat tissues contain less than 20 ngjmg of S-100 protein (Suzuki ef it.. 1982). The levels of S-100 protein in brain determined with the present method are slightly lower than the values (about iOO/cg/g rat brain) reported by Moore rf (II. (1968). Apparently low levels of S-100 protein in mouse adipose tissue and brain may due to a low cross-rea~ti~~ity of the mouse S-100 protein with the antibodies to beef S-100 protein, because the values were estimated with a beef S-100 protein as a standard and the assay system consisted of two steps of the specific immunoreaction with antibodies to beef S- I00 protein,
Table
Species Beef Rat Rabbit Human Pig Mouse
1. Levels of S-100 protein
Tissues Brain Adipose Brain Adipose Brain Adipose Adipose Adipose Brain Adipose
* Beef S-100 protein ** Mean + SD,
in brain
No. of samples I
tissue
1
tissue
5 5 1
tissue tissue tissue
6
tissue equivalents.
1 1 5 5
or cd.
The presence of S- 100 protein in the adipose tissues was confirmed by double immuI~odiffusion and immunoelectrophoresis of those crude extracts against the (anti-beef S-100 protein) serum. The concentrated extract and the antiserum were layered in the side wells and center wells. respectively (Fig. I ). The extract from beef or rat adipose tissue formed a clear single precipitin line against the antiserum. and the precipitin line fused with that produced between the standard beef S-100 protein and the antiserum. The extract from human or rabbit adipose tissue also formed a visible precipitin line against the antiserum, which fused with that of rat or beef adipose tissue extract. However, the concentrated (200-fold) extract of mouse and pig adipose tissue did not produce any detectable precipitin line against the antiserum. Immunoclectrophoresis of the extract of rat brain and adipose tissue against the anti-S-100 protein was shown in Fig. 2. Both extracts show a single precipitin arc with the antiserum at a similarly migrating position. These results are comparable to those obtained with the immunoassay, indicating the presence of high levels of S-100 protein in adipose tissues of several animal species. Developmental profiles of S-100 protein lecefs in rat Figure 3 shows a simultaneous determination of the development profiles of S-100 protein levels in rat brain and adipose tissue. Rats were fed ad libitum. A sharp increase in the level of brain S-100 protein was seen from 2 to 6 weeks after birth and the level reached a plateau after the 6th week. The levels at the 2nd and 6th week were 0.35 and 2.06~g~mg protein, respectively. The S-100 protein level in adipose tissue was determined at the 4. 6 and 10th week after birth. There is little abdominal adipose tissue in the 2-week aged rats. The S-100 protein levels in adipose tissue of the /l-week rat were about 0.4pgjmg and the levels were raised along with the rat growth. (1.1 and 1.6,~gjmg protein, at the 6th and 10th week, respectively.) Although S-100 protein in adipose tissue and central nervous tissue increases with a similar extent after the birth, the level of adipose S-100 protein might be modified by some dietary or hormonal
and adipose
(ng*/mg
tissue of various Soluble protein)
4850 1130 1980 ) 98** 1590 k 179 2590 222 82.6 f 55.4 149 482 + 180 2.90 & 0.69
animal
species
S-100 protein (ng*/g wet tissue) 80.0 2.15 47.6 * 2.1** 5.39 + 1.24 44.0 0.38 0.83 f 0.75 0.097 7.15 + 2.49 0.031 f 0.012
Fig. 1. Double immunodiffusion of brain and adipose tissue extracts of various animal species against a rabbit (anti-beef S-100) serum. Five microliters each of the concentrated extract were layered in the side wells [(a) rat brain containing 0.49 pg S-100 protein, calculated from the results of the immunoassay and concentration factor: (b) rat adipose tissue, 0.44 pg; (c) standard beef S-100, 0.53 pg: (d) beef adipose tissue. 0.44 pg: (e) beef brain, 0.88 pg; (f) human adipose tissue, 0. I I pg; (gt rabbit adipose tissue, 0.11 pg; (h) mouse adipose tissue. 0.003 iig: (i) pig adipose tissue. 0.002 pg]. The rabbit (anti-beef S-100) serum (5 ~1) was applied to the center wells.
Fig. 2. immunoelectrophoresis of rat brain protein) serum. Ten microliters each of the and 1mM EGTA were applied to the wells phoresis, 50 PI of the
and adipose tissue extracts against a rabbit (anti-beef S-100 concentrated extract containing about 0.5 itg S-100 protein [(a) brain extract; (b) adipose tissue extract]. After electroantiserum were applied to the trough.
611
S-100 protein in adipose
613
tissue
S-100 protein (0.222, 0.149 and 0.083 I.cg/mg, respectively). The S-100 protein level in adipose tisssue showed a developmental profile similar to that in the central nervous tissue. At 4th week after birth, S-100 protein levels in rat adipose tissue were about 0.4pg/ mg, and the levels at the 6th and 10th week were around 1.1 and I .6 pg/mg, respectively. REFERENCES
01
Age,
weeks
Fig. 3. Developmental profiles of S-100 protein levels in rat brain (t-0) and adipose tissue (A-A). The S-100 protein levels are expressed as beef S-100 protein equivalents/mg protein. Each point is a mean of 5 animals + SD.
factors as suggested previously (Suzuki et al., 1982), because the adipose levels were sharply increased after the weaning period. It is interesting to clarify the physiological role of S-100 protein in adipose tissues, which may bring us a basic understanding of unknown role(s) of this specific protein on the highly integrated function of the nervous system.
SUMMARY
The nervous system-specific S-100 protein was found at high levels, with an enzyme immunoassay, in adipose tissues of various animal species. The soluble S-100 protein levels in the tissues of beef and rat were 1.13 and 1.59 pg/mg protein, respectively, which were comparable to those of the central nervous tissues (4.85 and 1.98 pg/mg in beef and rat brains, respectively). Adipose tissues of rabbit, pig and human also contained relatively high levels of immunoreactive
BRAIIFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindlng. Amrlx1. Eioc/tern. 72, 248 -254. CALISSON P., MOORE B. W. & FRIESEN A. (1969) Eflect of calcium ion on S-100. a protein of the nervous system. Bioclwmistry 8, 43 18-4326. COCCHIA D. & MICIIETTI F. (1981) S-100 Antigen in satellite cells of the adrenal medulla and the superior cervical ganglion of the rat. Cc/l 7I.s.t. Rcs. 215, lO3- 112. COCCHIA D.. MICH~TTI F. & DONATO R. (1981) Immunochemical and immunocytochemical locahzation of S-100 antigen in normal human skin. Ntrtwc 294, X5-87. HAGLID K. G.. HA~SSO~; H. A. & Rijwn;icK L. (1977) S-100 in the central nervous system of rat. rabbit. and guinea pig during postnatal development. Brtrirl Rm. 122. 33 1~_345. KATO K.. NAKAJIMA T., ISHIC;URO Y. & MATSI’TANI T. (1982) Sensitive enzyme immunoassay for S-100 protein: Determination in human cerebrospinal Huid. BALMY/. Ras. 3, 2&28. MOORE B. W. (1975) Brain-specific proteins: S-100, 14-3-Z protein. and glial fibrillary protein. Atir. ~\‘c~troc/wm. I, I37 155. MOORE B. W., PEREZ V. J. & GEHRIX M. (1968) Assay and regional distribution of a soluble protein characteristic of the nervous system. J. Nruroclwrn. 15, 265-272. MOLLER M., IN~;LID A. & BOCK E. (1978) Immunohistochemica1 demonstration of S-100 protein and GFA protein in interstitial cells of rat pineal gland. Brclirt Ru. 140, l-13. NAKAJIMA T.. YAMAGUCHI H. & TAKAHASHI K. (1980) S-100 Protein in folliculostellate cells of the rat pituitary anterior lobe. Bruin Rrs. 191, 523-531. STLFANSSON K.. WOLLMANU R. L.. MOORE B. W. & ARNASON B. G. W. (1982) S-100 Protein in human chondrocytes. Natwe 295, 63-64. SUZUKI F.. NAKAJIMA T. & KATZ K. (1982) Peripheral distribution of nervous system-specific S-100 protein in rat. J. Biochern. 92, 835-838. UYEMURA K.. VINCENDOS G., COMBOS G. & MANDEL P. (1971) Purification and some properties of S-100 protein fractions from sheep and pig brains. J. Nrwoclwm. 18, 429-438.
0020-7 I I X;83/050609-05$03.00,0 Copyright 0 1983 Pergamon Press Ltd
1983
S-100 PROTEIN
IN ADIPOSE
TISSUE
K. KATCI’*, F. SUZUKI’ and T. NAKAJIMA*
I Department
of Biochemistry,
‘Pathology
Division,
Institute for Developmental Research, Aichi Prefectural Kasugai. Aichi 480-03, Japan and National Cancer Research Institute, Tokyo 104, Japan (Received
10 Auyust
1982)
Abstract-l. The nervous system-specific S-100 protein is present at high levels various animal species. 2. The adipose S-100 protein levels in beef and rat were 1.13 and 1.59 pg/mg which were comparable to the brain levels (beef, 4.85 pg/mg; rat. 1.98 pg/mg). 3. Adipose tissues of rabbit, pig and human contained 0 222, 0.149 and 0.083 respectively. 4. The S-100 protein level in rat adipose tissue showed a developmental profile brain.
INTRODUCTION
S-100 protein is a nervous system-specific protein, and it has been reported to be localized mainly in the cytoplasm of glial cells in nervous tissues (Moore, 1975). However, recent reports from several laboratories revealed immunocytochemically that the S-100 protein is also present in non-nervous tissues, such as interstitial cells of the pineal gland (Moller et al., 1978). satellite cells of the adrenal gland (Cocchia & Michetti. 1981), stellate cells of the hypophysis (Nakajima et ul., 1980), melanocytes and Langerhans cells of human skin (Cocchia ef al., 1981) and human chondrocytes (Stefansson et al., 1982). Recently, we have prepared a highly sensitive enzyme immunoassay system for S-100 protein (Kato et (I!., 1982). In researching the tissue distribution of S-100 protein with the immunoassay method, we found that rat adipose tissue and cartilage contained high levels of S-100 protein comparable to those in the central nervous tissue (Suzuki et al., 1982). In this report, we describe the levels of S-100 protein in adipose tissues from various animal species and the developmental profile of S-100 protein level in rat adipose tissue.
MATERIALS
AND
METHODS
Colony,
Emyme
immunoassay
in adipose protein,
tissues
of
respectively.
pg S-100 proteinimg, similar
to that in the
fbr S- IO0 protein
The enzyme immunoassay system for S-100 protein was composed of a silicone rubber solid-phase (413 x 4 mm) with immobilized (anti-beef S-100) antibodies and the antibodies labelled with p-D-galactosidase from Escherickia coli as described recently (Kato et al., 1982). Samples containing S-100 protein were incubated at 30°C for 5 hr with a piece of the solid-phase, and then the solid-phase with bound S-100 protein was reacted with the enzyme-labelled antibodies at 4°C overnight. From the fi-o-galactosidase activity bound on the solid-phase, amounts of S-100 protein could be determined with a minimum detectable sensitivity of 3 pg beef S-100 protein (Kato ct al., 1982). Standard
S- 100 protein
Immunologically, S-100 protein is a species-nonspecific protein, and the antiserum to beef S-100 protein crossreacts with S-100 proteins from various animal species (Moore, 1975). Therefore, we have determined the S-100 protein levels in brains and adipose tissues from various animals with the above immunoassay system by use of the purified beef S-100 protein as a standard. and the results are expressed as pg or ng of beef S-100 protein equiva-
lents/mg protein or g wet weight. Concentrations of the purified S-100 protein were estimated by using a value E:& = 3.44 (Calisson et al., 1969). Protein concentrations of the extract were determined with Bio-Rad Protein Assay (Bio-Rad), which utilizes a principle of protein-dye binding (Bradford, 1976). Tissue extract
S-100 protein was purified from beef brain by the method of Uyemura et al. (1971) and the final preparation was electrophoretically pure as described previously (Nakajima et al.. 1980). The antiserum against S-100 protein was raised in New Zealand white rabbits by injecting the purificd S-100 protein with methylated bovine serum albumin ~1sdescribed by Haglid ct al. (1977). The antiserum was found to be specific to S-100 protein showing a single precipitin line on double immunodiffusion against a crude extract of beef brain (Fig. 1).
*TO whom correspondence should be addressed: Dr Kanefusa Kate. Department of Biochemistry, Institute for Aichi Prefectural Colony. Developmental Research. Kamiya. Kasugai. Aichi 4X&03. Japan.
Fresh brains and abdominal adipose tissues of beef and pig were obtained from a slaughter house. Female rats (Wistar) aged 2-10 weeks, male adult mice (129/SvJ) and a male rabbit were sacrificed and whole brains and abdomlnal adipose tissues were quickly removed. Human tissues were obtained on the autopsy. All samples were kept frozen at -80°C until analysis. The frozen tissues were homogenized at 0°C in 4-vol. (v/w) of 0.05 M Tris-HCI buffer, pH 7.0, containing 0.1 M NaCl with a glass homogenizer. The homogenate was centrifuged at 4-C at 107,OOOg for 1 hr, and the supernatant was used for the assay of S-100 protein. Each extract was variously diluted (50,00&50-fold) with 0.01 M sodium phosphate buffer, pH 7.0 containing 0.3 M NaCI, 1 mM MgCl*, 1 mM calcium acetate, O.l”,, bovine serum albumin (Armour Phar-
610
K. KATO
maceuticais). OS”,, gelatin (Difco Laboratories) and O.l”,, NaN, at room temperature. Aiiquots (0.5 ml) of the diluted samples were subjected in duplicate to the immunoassay as described above.
Doubic ~mmunodiffusion of the crude extract against the antiserum to beef S-100 protein was performed at room temperature (l&)-25’C) on a slide piass (2.5 x 7 cm) covered w& 3 ml of l”,, agarose containing 0.9”, NaCI, I mM calcium acetate and 20mM Tris HCI buffer, aH 7.0. After variously concentrated (i&200-fold with M’inicon B-15. Amicon Corp.). 5 iti each of the crude extract were applied on the wells. Immunoeiectrophoresis was carried out on the slide glass covered with 3 ml of i.2”,, agarose in 60 mM barbital buffer. pH 8.6 at 4 C. Run time was 4t?min at 3 mA/slide glass. After eiectrophoresis. anti-S-100 serum was applied to the trough at room temperature. Precipitin lines were observed after 24 hr.
RESVLTS AUD
DISCUSSION
Lecels cf S-l 011 grofein in adipose animal species
tissues qf‘ cariaus
The immunoassay of S-100 protein in crude extracts was performed with the variously diluted samples at two or three different concentrations of each extract, and the results were expressed as means of the assays. As shown in Table 1, S-100 protein is present in adipose tissues of beef and rat at high levels comparable to those of central nervous tissues when compared on the basis of mg protein. Adipose tissues of rabbit, pig and human also contain considerably high levels of S-100 protein, since most of rat tissues contain less than 20 ngjmg of S-100 protein (Suzuki ef it.. 1982). The levels of S-100 protein in brain determined with the present method are slightly lower than the values (about iOO/cg/g rat brain) reported by Moore rf (II. (1968). Apparently low levels of S-100 protein in mouse adipose tissue and brain may due to a low cross-rea~ti~~ity of the mouse S-100 protein with the antibodies to beef S-100 protein, because the values were estimated with a beef S-100 protein as a standard and the assay system consisted of two steps of the specific immunoreaction with antibodies to beef S- I00 protein,
Table
Species Beef Rat Rabbit Human Pig Mouse
1. Levels of S-100 protein
Tissues Brain Adipose Brain Adipose Brain Adipose Adipose Adipose Brain Adipose
* Beef S-100 protein ** Mean + SD,
in brain
No. of samples I
tissue
1
tissue
5 5 1
tissue tissue tissue
6
tissue equivalents.
1 1 5 5
or cd.
The presence of S- 100 protein in the adipose tissues was confirmed by double immuI~odiffusion and immunoelectrophoresis of those crude extracts against the (anti-beef S-100 protein) serum. The concentrated extract and the antiserum were layered in the side wells and center wells. respectively (Fig. I ). The extract from beef or rat adipose tissue formed a clear single precipitin line against the antiserum. and the precipitin line fused with that produced between the standard beef S-100 protein and the antiserum. The extract from human or rabbit adipose tissue also formed a visible precipitin line against the antiserum, which fused with that of rat or beef adipose tissue extract. However, the concentrated (200-fold) extract of mouse and pig adipose tissue did not produce any detectable precipitin line against the antiserum. Immunoclectrophoresis of the extract of rat brain and adipose tissue against the anti-S-100 protein was shown in Fig. 2. Both extracts show a single precipitin arc with the antiserum at a similarly migrating position. These results are comparable to those obtained with the immunoassay, indicating the presence of high levels of S-100 protein in adipose tissues of several animal species. Developmental profiles of S-100 protein lecefs in rat Figure 3 shows a simultaneous determination of the development profiles of S-100 protein levels in rat brain and adipose tissue. Rats were fed ad libitum. A sharp increase in the level of brain S-100 protein was seen from 2 to 6 weeks after birth and the level reached a plateau after the 6th week. The levels at the 2nd and 6th week were 0.35 and 2.06~g~mg protein, respectively. The S-100 protein level in adipose tissue was determined at the 4. 6 and 10th week after birth. There is little abdominal adipose tissue in the 2-week aged rats. The S-100 protein levels in adipose tissue of the /l-week rat were about 0.4pgjmg and the levels were raised along with the rat growth. (1.1 and 1.6,~gjmg protein, at the 6th and 10th week, respectively.) Although S-100 protein in adipose tissue and central nervous tissue increases with a similar extent after the birth, the level of adipose S-100 protein might be modified by some dietary or hormonal
and adipose
(ng*/mg
tissue of various Soluble protein)
4850 1130 1980 ) 98** 1590 k 179 2590 222 82.6 f 55.4 149 482 + 180 2.90 & 0.69
animal
species
S-100 protein (ng*/g wet tissue) 80.0 2.15 47.6 * 2.1** 5.39 + 1.24 44.0 0.38 0.83 f 0.75 0.097 7.15 + 2.49 0.031 f 0.012
Fig. 1. Double immunodiffusion of brain and adipose tissue extracts of various animal species against a rabbit (anti-beef S-100) serum. Five microliters each of the concentrated extract were layered in the side wells [(a) rat brain containing 0.49 pg S-100 protein, calculated from the results of the immunoassay and concentration factor: (b) rat adipose tissue, 0.44 pg; (c) standard beef S-100, 0.53 pg: (d) beef adipose tissue. 0.44 pg: (e) beef brain, 0.88 pg; (f) human adipose tissue, 0. I I pg; (gt rabbit adipose tissue, 0.11 pg; (h) mouse adipose tissue. 0.003 iig: (i) pig adipose tissue. 0.002 pg]. The rabbit (anti-beef S-100) serum (5 ~1) was applied to the center wells.
Fig. 2. immunoelectrophoresis of rat brain protein) serum. Ten microliters each of the and 1mM EGTA were applied to the wells phoresis, 50 PI of the
and adipose tissue extracts against a rabbit (anti-beef S-100 concentrated extract containing about 0.5 itg S-100 protein [(a) brain extract; (b) adipose tissue extract]. After electroantiserum were applied to the trough.
611
S-100 protein in adipose
613
tissue
S-100 protein (0.222, 0.149 and 0.083 I.cg/mg, respectively). The S-100 protein level in adipose tisssue showed a developmental profile similar to that in the central nervous tissue. At 4th week after birth, S-100 protein levels in rat adipose tissue were about 0.4pg/ mg, and the levels at the 6th and 10th week were around 1.1 and I .6 pg/mg, respectively. REFERENCES
01
Age,
weeks
Fig. 3. Developmental profiles of S-100 protein levels in rat brain (t-0) and adipose tissue (A-A). The S-100 protein levels are expressed as beef S-100 protein equivalents/mg protein. Each point is a mean of 5 animals + SD.
factors as suggested previously (Suzuki et al., 1982), because the adipose levels were sharply increased after the weaning period. It is interesting to clarify the physiological role of S-100 protein in adipose tissues, which may bring us a basic understanding of unknown role(s) of this specific protein on the highly integrated function of the nervous system.
SUMMARY
The nervous system-specific S-100 protein was found at high levels, with an enzyme immunoassay, in adipose tissues of various animal species. The soluble S-100 protein levels in the tissues of beef and rat were 1.13 and 1.59 pg/mg protein, respectively, which were comparable to those of the central nervous tissues (4.85 and 1.98 pg/mg in beef and rat brains, respectively). Adipose tissues of rabbit, pig and human also contained relatively high levels of immunoreactive
BRAIIFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindlng. Amrlx1. Eioc/tern. 72, 248 -254. CALISSON P., MOORE B. W. & FRIESEN A. (1969) Eflect of calcium ion on S-100. a protein of the nervous system. Bioclwmistry 8, 43 18-4326. COCCHIA D. & MICIIETTI F. (1981) S-100 Antigen in satellite cells of the adrenal medulla and the superior cervical ganglion of the rat. Cc/l 7I.s.t. Rcs. 215, lO3- 112. COCCHIA D.. MICH~TTI F. & DONATO R. (1981) Immunochemical and immunocytochemical locahzation of S-100 antigen in normal human skin. Ntrtwc 294, X5-87. HAGLID K. G.. HA~SSO~; H. A. & Rijwn;icK L. (1977) S-100 in the central nervous system of rat. rabbit. and guinea pig during postnatal development. Brtrirl Rm. 122. 33 1~_345. KATO K.. NAKAJIMA T., ISHIC;URO Y. & MATSI’TANI T. (1982) Sensitive enzyme immunoassay for S-100 protein: Determination in human cerebrospinal Huid. BALMY/. Ras. 3, 2&28. MOORE B. W. (1975) Brain-specific proteins: S-100, 14-3-Z protein. and glial fibrillary protein. Atir. ~\‘c~troc/wm. I, I37 155. MOORE B. W., PEREZ V. J. & GEHRIX M. (1968) Assay and regional distribution of a soluble protein characteristic of the nervous system. J. Nruroclwrn. 15, 265-272. MOLLER M., IN~;LID A. & BOCK E. (1978) Immunohistochemica1 demonstration of S-100 protein and GFA protein in interstitial cells of rat pineal gland. Brclirt Ru. 140, l-13. NAKAJIMA T.. YAMAGUCHI H. & TAKAHASHI K. (1980) S-100 Protein in folliculostellate cells of the rat pituitary anterior lobe. Bruin Rrs. 191, 523-531. STLFANSSON K.. WOLLMANU R. L.. MOORE B. W. & ARNASON B. G. W. (1982) S-100 Protein in human chondrocytes. Natwe 295, 63-64. SUZUKI F.. NAKAJIMA T. & KATZ K. (1982) Peripheral distribution of nervous system-specific S-100 protein in rat. J. Biochern. 92, 835-838. UYEMURA K.. VINCENDOS G., COMBOS G. & MANDEL P. (1971) Purification and some properties of S-100 protein fractions from sheep and pig brains. J. Nrwoclwm. 18, 429-438.