Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Molecular weight forms of corneal aldehyde dehydrogenase Tjahjono D. Gondhowiardjo, Nicolaas J. van Haeringen & Aize Kijlstra To cite this article: Tjahjono D. Gondhowiardjo, Nicolaas J. van Haeringen & Aize Kijlstra (1992) Molecular weight forms of corneal aldehyde dehydrogenase, Current Eye Research, 11:4, 377-381, DOI: 10.3109/02713689209001791 To link to this article: http://dx.doi.org/10.3109/02713689209001791
Published online: 02 Jul 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [Imperial College London Library]
Date: 02 April 2016, At: 15:38
Current Eve Rksearch
SHORT COMMUNICATION Volume I I number 4 1992. 377-381
Molecular weight forms of corneal aldehyde dehydrogenase
Tjahjono D.Gondhowiardjo, Nicolaas J.van Haeringen' and Aize Kijlstra2
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Department of Ophthalmology, University of Indonesia, Jakarta, Indonesia, 'The Netherlands Ophthalmic Research Institute and 2Department of Ophthalmology, University of Amsterdam, The Netherlands
ABSTRACT Aldehyde dehydrogenase has recently been shown t o be one of the major soluble proteins in the mammalian cornea. The enzyme has a subunit molecular weight of 54 kd and gel filtration experiments indicate that a dimer molecule is the enzymatically active species. The purpose of the studies described here was to investigate whether oligomeric forms of this enzyme could also be detected using the much faster SDS-PAGE mini-gel electrophoresis technique combined with immunoblotting and "in gel" enzyme detection. Low temperature treatment of samples prior to electrophoresis revealed that both human and bovine corneal ALDH are mainly present as a 5 4 kd and as a dimer molecule with an apparent molecular weight of 88 kd. Bovine corneal ALDH also contained larger oligomers with a molecular weight of 1 10, 154 and 21 0 kd respectively. The classical 3 minutes boiling procedure prior to SDSPAGE dissociated the oligomers into the 54 kd subunit. Zymography experiments showed that enzyme activity was only present in the 88 kd form of corneal ALDH. Pretreatment of corneal ALDH at various temperatures showed that the temperature induced shift of the 8 8 kd species to the 5 4 kd subunit parallelled the decrease in enzymatic activity. The fact that reduction of samples with DTT did not dissociate the 8 8 kd form suggests that disulfide bridge formation is not involved in the oligomerisation of corneal ALDH.
Recently, we have found that human corneal extracts contained the same enzyme. Moreover, we found that the enzyme activity in the human corneal epithelium was almost three times greater than in the corneal stroma, whereas the endothelium showed very little enzyme activity (18). The variable substrate preference found for corneal ALDH in various mammalian species indicates that it's enzymatic activity is not strictly conserved during evolution, indicating a structural role for this protein in the cornea (1-3,19,20). ALDH is not only present in the eye, but has also been detected in various other organs (2.2 1-23). In the human and horse liver the protein is present as a tetramer, whereby the subunit size was shown t o be 54 kd (22.23). Gel filtration experiments indicate that the enzymatically active corneal ALDH was a dimer (1,2,19). We reinvestigated the oligomeric corneal ALDH forms using an SDS-PAGE mini-gel electrophoresis system and visualized the protein bands by a combination of either Coomassie or Silver staining, immunoblotting and zymography techniques. Preparation of corneal ALDH, BCP-54 antibody,
Aldehyde dehydrogenase has recently been identified as the major soluble protein of the bovine cornea ( 1 ). High levels of
electrophoretic techniques, immunoblotting, the aldehyde dehydrogenase assay and zymography of ALDH have been
ocular aldehyde dehydrogenase (ALDHI activity have now been
described extensively elsewhere (18). Elution of SDS from the gel
reported in several mammalian species. Within the eye, ALDH is
after SDS-PAGE electrophoresis was done using modification
not only detected in the cornea but has also been described in
from the technique described by Birkedal-Hansen (241, the gel
the lens and retina (2,3). The corneal aldehyde dehydrogenase is identical to a
being washed with 20 ml 2.5% Triton X-1 00 (BDH Chemicals LTD, Poole, England) in distilled water for 40 min., 2 0 ml 2.5 %
8corneal protein which was earlier described extensively by a
Triton X-100 in 0.1 M TRlS pH 8.0 (40 min) and 2 0 ml 0.1 M
number of groups (4-12) and which is known as bovine corneal
TRlS pH 8.0, 1 m M EDTA (E. Merck, Darmstadt, FRG) 1 m M
protein 5 4 (BCP 54). A possible role of this protein as an auto-
DTT (Bio-Rad, Richmond, USA) for 1 hour. Wash-out procedures
antigen in the pathogenesis of corneal diseases but also in uveitis
were done under agitation at room temperature.
has been reported ( 13-16).lmmunohistochemical studies have
The effect of temperature treatment of the sample on the
shown that it is present in the corneal epithelium, the stromal
migration o f the bovine corneal ALDH after SDS-PAGE is shown
keratocytes, the endothelium and the epithelial cells of the
in fig.1. With the normal procedure, treatment of the sample at
anterior capsule of the lens (17).
100" C for three minutes resulted in a heavy 5 4 kd band. On the
Received on Deccniher 2. 1991; accepted on March 13, 1992
0 Oxford University Press
377
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Current Eye Research
Fig. 1. Effect of sample treatment temperature on the molecular weight forms of bovine corneal ALDH after SDS-PAGE in the presence of reducing agents. Samples ( 1 .spa protein) were placed in a waterbath and treated
at various temperatures ranging from room temperature to 100" C for three minutes before application onto the gels. (staining: Coomassie blue).
Fig.2. Detection of corneal ALDH molecular weioht forms with immunoblotting using a rabbit anti bovine corneal ALDH antibody.
contrary, non heated samples revealed several bands, with
C resulted in a decrease of the 88 kd band and a concomitant
molecular weights of 54, 88, 110, 154 and 210 kd respectively.
increase of the 54 kd band. The 154 kD and 210 kD bands
Increasing the pretreatment temperature of the sample above 40"
decreased a t temperatures above 70" C. In the absence of
378
Current Eye Research
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
a
b
ALDH activity Iu/rng
I
%ofthe88kD
351
Fig.3. SDS-PAGE of human corneal ALDH (0.05 p g protein) in the presence of reducing agents. Unheated sample (lane 11 and heat (100" C) pretreated sample (lane 2). The left lane contains molecular weight markers. (Silver staining).
reducing agents the effect of temperature on the SDS-PAGE protein patterns was identical to that shown in fig.1, which was performed under reducing conditions. lmmunoblotting of a similar gel as shown above, using a rabbit anti bovine corneal protein 54 kd antibody showed that all
temp.
OC
the bands previously shown in fig. 1 stained with this antibody. After heating the sample for 3 min. at 100" C all irnmunoreactivity was present in one 5 4 kD band (fig.2). With the temperature increase the immunostaining of the 54 kd band became heavier. SDS-PAGE of purified human corneal ALDH also showed
Fig.4a. Aldehyde dehydrogenase enzyme activity profile of bovine corneal ALDH after pre-incubation for 3 min. at various temperatures. Results show the mean f 1 SD of five samples. Fig.4b. Effect of sample treatment temperature on the percentage of the 88 kD band compared to the total protein, as measured by scanning densitometry. Staining of the gels was performed with Coomassie blue.
that pretreatment of samples at room temperature resulted in t w o major bands of 88 and 54 kd. Pretreatment of the sample at
100" C resulted in the disappearance of the 88 kd band and in an increased intensity of the 5 4 kd protein (fig.31. The effect of temperature on bovine ALDH activity
(fig.4a) shows that the activity increases when the protein is preincubated a t 30" C for three minutes as compared to room temperature. Higher pretreatment temperatures resulted in a
379
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Current Eye Research
Fig.5. SDS-PAGE of bovine corneal extract (2.5 pg protein) under reduced (lane 2) and non-reduced condition (lane 3) visualized with Coomassie staining. Lane 1 contains molecular weight
declining enzymatic activity. No more enzyme activity could be
markers. Lane 4 and 5 are identical t o lane 2 and 3 but were stained for ALDH activity (zymogram) after elution of SDS with Triton X-1 00.
In this study we thus show that SDS-PAGE analysis of
measured when ALDH had been pretreated at 60" C. Using a
purified bovine and human corneal aldehyde dehydrogenase
human corneal epithelial extract the same temperature
preparations revealed various molecular forms with a subunit size
dependency on ALDH activity was observed (data not shown).
of 5 4 kd. We confirmed the thermolability of corneal ALDH (19)
The effect of temperature on the intensity of the 88 kd
and found that enzyme activity is harboured in the 88 kd ALDH
band after SDS-PAGE of purified bovine ALDH as measured with
species. Preincubation of ALDH at high temperatures results in
scanning densitometry showed that it parallelled the decrease in
one protein with a molecular weight of 54 kd.
enzymatic activity (fig.4b). Visualization of aldehyde dehydrogenase activity in a
Sephadex gel filtration of corneal ALDH performed by Evces (19) and Abedinia (1) indicated the presence of a dimer
bovine corneal epithelial extract on the SDS-PAGE gel after the
molecule. This so-called dimer may be identical to the 88 kd
elution of SDS with Triton X-100 revealed that the activity of
protein reported in the present study. The apparent molecular
corneal aldehyde dehydrogenase was associated only with the 88
weight is lower than would be expected. On the other hand
kd band (fig.5). Enzyme activity in the zymograms was not
dimerisation could lead t o conformational changes resulting in an
affected by prior reduction of the samples with DTT. SDS-PAGE
apparent smaller complex than expected in SDS-PAGE analysis.
zymograms of 7 individual human corneal epithelial extracts also
This is the first report showing that the activity of corneal
revealed that the enzyme activity was always present in the 88
ALDH on an SDS-PAGE gel using zymography technique is
kd band (data not shown).
associated with the 88 kd species. This report also confirms the
Current Eye Research observation that the heat lability of corneal ALDH is correlated with a shift of the 88 kd species to the 54 kd form. ACKNOWLEDGEMENTS T.D.G gratefully acknowledge the Hoornvlies Stichting Nederlands and the Nelly Reef Fund for their scholarship.
CORRESPONDING AUTHOR Prof.Dr. A. Kijlstra, Dept. Ophthalmo-Immunology, The Netherlands Ophthalmic Research Institute. P.0.Box 12141
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
1100 A.C. Amsterdam Z.O.The Netherlands.
REFERENCES 1. Abedinia, M., Pain, T., Algar, E.M. and Holmes, R.S.(1990) Bovine corneal aldehyde dehydrogenase: the major soluble corneal protein with a possible dual protective role for the eye. Exp. Eye Res. 419-426. 2. Holmes, R.S. and VandeBerg, J.L.(1986) Ocular NAD-dependent Alcohol dehydrogenase and Aldehyde 383-396. dehydrogenase in the baboon. Exp. Eye Res. 3. Holmes, R.S.(1988) Alcohol dehydrogenases and aldehyde dehydrogenases of anterior eye tissues from humans and other mammals. In "Biomedical and Social Aspects of Alcohol and alcoholism" (eds. Kuriyama, K., Takada, A., and Ishii, H.). Pp. 51-57, Elsevier Science Publisher, Amsterdam, The Netherlands. 4. Alexander, R.J., Silverman, B. and Henley, W.L.(1981) Isolation and characterization of BCP 54, the major soluble protein of bovine cornea. Exp.Eye Res. 2 205-216. 5. Silverman, B., Alexander, R.J. and Henley, W.L.(1981) Tissue and species specificity of BCP 54, the major soluble 19-29. protein of bovine cornea. Exp. Eye Res. 6. Kruit, P.J., Broersma, L., v.d.Gaag, R. and Kijlstra, A.( 1986) Clinical and experimental studies concerning circulating antibodies t o corneal epithelium antigens. Doc. Ophthalmol. & 43-51. 7. Kong, A.S., Henley, W.L. and Luntz, M.H. (1988) lntracellular distribution of corneal protein BCP 5 4 in epithelial cells. In "World Uveitis Symposium" (Eds. Belfort Jr, R., Petrilli A.M.N and Nussenblatt, R.), Pp. 109-1 16. Proceeding of the first world uveitis symposium held in Guaruja' Sao Paulo, Brazil. 8 . Cooper, D.L., Baptist, E.W., Lee, H., Isola, N. and Klintworth, G. K. (1990) Partial aminoacid sequence determination of Bovine Corneal Protein 54 K (BCP 54). Curr. Eye Res. 9.781 -786. 9. Verjans, G.M.G.M., Verhagen, C., Hoeckzema, R. and Kijlstra, A.(1990) Bovine corneal protein 54 (BCP 54) shows strong homology to rat tumor-associated aldehyde dehydrogenase (T-ALDH). Letter to the Editor. Curr. Eye Res. 3, 1217-1218. 10. Cooper, D.L. (1990) Response t o the Editors. Curr. Eye Res. 3, 1219-1220. 11. Cooper, D.L., Baptist, E.W., Enghild, J.J., Isola, N.R. and Klinworth, G.K. (1991 1 Bovine corneal protein 54K (BCP54) is a homologue of the tumor associated(c1ass 3) rat aldehyde dehydrogenase (RATALD).Gene, 98, 201 -207. 12. Verhagen, C.,Hoekzema, R., Verjans, G.M.G.M. and Kijlstra, A. (19911 Identification of bovine corneal protein 5 4 (BCP 54) as an aldehyde dehydrogenase. Letter to the Editor. Exp. 283-284. Eye Res.
a
a,
13. Kruit, P.J., v.d.Gaag, R., Broersma, L. and Kijlstra, A.(1986) Autoimmunity against corneal antigens. I. Isolation of a soluble 5 4 kD corneal epithelium antigen. Curr. Eye Res. 5, 31 3-320. 14. Kruit, P.J., Broersma. L., v.d.Gaag, R. and Kijlstra, A.(1986) Clinical and experimental studies concerning circulating antibodies to corneal epithelium antigens. Doc.Ophthalrnol. 43-51. 15. Kong, A.S., Henley, W.L. and Luntz, M.H. (1989) Longitudinal study of serum antibody response to bovine corneal protein (BCP 54) in Behcet's Disease. Ophthalmic Re$. 401-405. 16. v.d.Gaag, R., Broersma, L., Rothova, A., Baarsma, S. and Kijlstra, A.(1989) Immunity to a corneal antigen in Fuchs' heterochromic cyclitis patients. Invest. Ophthalmol. Vis. Sci. ;1IL 443-448. 17. Eype, A.A., Kruit, P.J., v der Gaag, R., Neuteboom, G.H.G., Broersma, L. and Kijlstra, A.(1987) Autoimmunity against corneal antigens. II. Accessibility of the 54 kD corneal antigen for circulating antibodies. Curr. Eye Res. 6, 467-475. 18. Gondhowiardjo, T.D. v Haeringen, N.J, Hoekzema, R, Pels, L and Kijlstra, A (1991 1 Detection of aldehyde dehydrogenase activity in human corneal extracts. Curr. Eye Res. 1(L 10011007. 19. Evces, S. and Lindahl, R. (1989) Characterization of rat cornea aldehyde dehydrogenase. Arch. Biochem. Biophy. 274. 518-524. 20. Messiha, F.S. (1981 1 Species-dependent ocular aldehyde dehydrogenase. Res. Comm. Chem. Pathol. Pharmacol. 365-368. 21. Harada, S., Agarwal, D.P. and Werner-Goedde, H.(1980) Electrophoretic and biochemical studies of human aldehyde dehydrogenase isoenzymes in various tissues. Life Sciences, 1773-1 780. 22. Greenfield, N.J. and Pietruszko, R. (1977) Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes. 35-45. Biochim. Biophys. Acta, 23. Hempel, J., v Bahr-Lindstrbm, H. and Jbrnvall, H. (1984) Aldehyde dehydrogenase from human liver. Primary structure of the cytoplasmic isoenzyme. Eur. J. Biochem. 14L.21-35. 24. Birkedal-Hansen, H (1987) Catabolism and turnover of collagens: Collagenases. In "Methods in Enzymology" vol. 144, Structural and contractile proteins.(Ed. Cunningham, L.W) Pp.140-161. Academic Press, Inc., Orlando, San Diego, New York, Austin, Boston, London, Sidney, Tokyo, Toronto.
m,
u,
38 1
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Molecular weight forms of corneal aldehyde dehydrogenase Tjahjono D. Gondhowiardjo, Nicolaas J. van Haeringen & Aize Kijlstra To cite this article: Tjahjono D. Gondhowiardjo, Nicolaas J. van Haeringen & Aize Kijlstra (1992) Molecular weight forms of corneal aldehyde dehydrogenase, Current Eye Research, 11:4, 377-381, DOI: 10.3109/02713689209001791 To link to this article: http://dx.doi.org/10.3109/02713689209001791
Published online: 02 Jul 2009.
Submit your article to this journal
Article views: 5
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [Imperial College London Library]
Date: 02 April 2016, At: 15:38
Current Eve Rksearch
SHORT COMMUNICATION Volume I I number 4 1992. 377-381
Molecular weight forms of corneal aldehyde dehydrogenase
Tjahjono D.Gondhowiardjo, Nicolaas J.van Haeringen' and Aize Kijlstra2
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Department of Ophthalmology, University of Indonesia, Jakarta, Indonesia, 'The Netherlands Ophthalmic Research Institute and 2Department of Ophthalmology, University of Amsterdam, The Netherlands
ABSTRACT Aldehyde dehydrogenase has recently been shown t o be one of the major soluble proteins in the mammalian cornea. The enzyme has a subunit molecular weight of 54 kd and gel filtration experiments indicate that a dimer molecule is the enzymatically active species. The purpose of the studies described here was to investigate whether oligomeric forms of this enzyme could also be detected using the much faster SDS-PAGE mini-gel electrophoresis technique combined with immunoblotting and "in gel" enzyme detection. Low temperature treatment of samples prior to electrophoresis revealed that both human and bovine corneal ALDH are mainly present as a 5 4 kd and as a dimer molecule with an apparent molecular weight of 88 kd. Bovine corneal ALDH also contained larger oligomers with a molecular weight of 1 10, 154 and 21 0 kd respectively. The classical 3 minutes boiling procedure prior to SDSPAGE dissociated the oligomers into the 54 kd subunit. Zymography experiments showed that enzyme activity was only present in the 88 kd form of corneal ALDH. Pretreatment of corneal ALDH at various temperatures showed that the temperature induced shift of the 8 8 kd species to the 5 4 kd subunit parallelled the decrease in enzymatic activity. The fact that reduction of samples with DTT did not dissociate the 8 8 kd form suggests that disulfide bridge formation is not involved in the oligomerisation of corneal ALDH.
Recently, we have found that human corneal extracts contained the same enzyme. Moreover, we found that the enzyme activity in the human corneal epithelium was almost three times greater than in the corneal stroma, whereas the endothelium showed very little enzyme activity (18). The variable substrate preference found for corneal ALDH in various mammalian species indicates that it's enzymatic activity is not strictly conserved during evolution, indicating a structural role for this protein in the cornea (1-3,19,20). ALDH is not only present in the eye, but has also been detected in various other organs (2.2 1-23). In the human and horse liver the protein is present as a tetramer, whereby the subunit size was shown t o be 54 kd (22.23). Gel filtration experiments indicate that the enzymatically active corneal ALDH was a dimer (1,2,19). We reinvestigated the oligomeric corneal ALDH forms using an SDS-PAGE mini-gel electrophoresis system and visualized the protein bands by a combination of either Coomassie or Silver staining, immunoblotting and zymography techniques. Preparation of corneal ALDH, BCP-54 antibody,
Aldehyde dehydrogenase has recently been identified as the major soluble protein of the bovine cornea ( 1 ). High levels of
electrophoretic techniques, immunoblotting, the aldehyde dehydrogenase assay and zymography of ALDH have been
ocular aldehyde dehydrogenase (ALDHI activity have now been
described extensively elsewhere (18). Elution of SDS from the gel
reported in several mammalian species. Within the eye, ALDH is
after SDS-PAGE electrophoresis was done using modification
not only detected in the cornea but has also been described in
from the technique described by Birkedal-Hansen (241, the gel
the lens and retina (2,3). The corneal aldehyde dehydrogenase is identical to a
being washed with 20 ml 2.5% Triton X-1 00 (BDH Chemicals LTD, Poole, England) in distilled water for 40 min., 2 0 ml 2.5 %
8corneal protein which was earlier described extensively by a
Triton X-100 in 0.1 M TRlS pH 8.0 (40 min) and 2 0 ml 0.1 M
number of groups (4-12) and which is known as bovine corneal
TRlS pH 8.0, 1 m M EDTA (E. Merck, Darmstadt, FRG) 1 m M
protein 5 4 (BCP 54). A possible role of this protein as an auto-
DTT (Bio-Rad, Richmond, USA) for 1 hour. Wash-out procedures
antigen in the pathogenesis of corneal diseases but also in uveitis
were done under agitation at room temperature.
has been reported ( 13-16).lmmunohistochemical studies have
The effect of temperature treatment of the sample on the
shown that it is present in the corneal epithelium, the stromal
migration o f the bovine corneal ALDH after SDS-PAGE is shown
keratocytes, the endothelium and the epithelial cells of the
in fig.1. With the normal procedure, treatment of the sample at
anterior capsule of the lens (17).
100" C for three minutes resulted in a heavy 5 4 kd band. On the
Received on Deccniher 2. 1991; accepted on March 13, 1992
0 Oxford University Press
377
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Current Eye Research
Fig. 1. Effect of sample treatment temperature on the molecular weight forms of bovine corneal ALDH after SDS-PAGE in the presence of reducing agents. Samples ( 1 .spa protein) were placed in a waterbath and treated
at various temperatures ranging from room temperature to 100" C for three minutes before application onto the gels. (staining: Coomassie blue).
Fig.2. Detection of corneal ALDH molecular weioht forms with immunoblotting using a rabbit anti bovine corneal ALDH antibody.
contrary, non heated samples revealed several bands, with
C resulted in a decrease of the 88 kd band and a concomitant
molecular weights of 54, 88, 110, 154 and 210 kd respectively.
increase of the 54 kd band. The 154 kD and 210 kD bands
Increasing the pretreatment temperature of the sample above 40"
decreased a t temperatures above 70" C. In the absence of
378
Current Eye Research
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
a
b
ALDH activity Iu/rng
I
%ofthe88kD
351
Fig.3. SDS-PAGE of human corneal ALDH (0.05 p g protein) in the presence of reducing agents. Unheated sample (lane 11 and heat (100" C) pretreated sample (lane 2). The left lane contains molecular weight markers. (Silver staining).
reducing agents the effect of temperature on the SDS-PAGE protein patterns was identical to that shown in fig.1, which was performed under reducing conditions. lmmunoblotting of a similar gel as shown above, using a rabbit anti bovine corneal protein 54 kd antibody showed that all
temp.
OC
the bands previously shown in fig. 1 stained with this antibody. After heating the sample for 3 min. at 100" C all irnmunoreactivity was present in one 5 4 kD band (fig.2). With the temperature increase the immunostaining of the 54 kd band became heavier. SDS-PAGE of purified human corneal ALDH also showed
Fig.4a. Aldehyde dehydrogenase enzyme activity profile of bovine corneal ALDH after pre-incubation for 3 min. at various temperatures. Results show the mean f 1 SD of five samples. Fig.4b. Effect of sample treatment temperature on the percentage of the 88 kD band compared to the total protein, as measured by scanning densitometry. Staining of the gels was performed with Coomassie blue.
that pretreatment of samples at room temperature resulted in t w o major bands of 88 and 54 kd. Pretreatment of the sample at
100" C resulted in the disappearance of the 88 kd band and in an increased intensity of the 5 4 kd protein (fig.31. The effect of temperature on bovine ALDH activity
(fig.4a) shows that the activity increases when the protein is preincubated a t 30" C for three minutes as compared to room temperature. Higher pretreatment temperatures resulted in a
379
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
Current Eye Research
Fig.5. SDS-PAGE of bovine corneal extract (2.5 pg protein) under reduced (lane 2) and non-reduced condition (lane 3) visualized with Coomassie staining. Lane 1 contains molecular weight
declining enzymatic activity. No more enzyme activity could be
markers. Lane 4 and 5 are identical t o lane 2 and 3 but were stained for ALDH activity (zymogram) after elution of SDS with Triton X-1 00.
In this study we thus show that SDS-PAGE analysis of
measured when ALDH had been pretreated at 60" C. Using a
purified bovine and human corneal aldehyde dehydrogenase
human corneal epithelial extract the same temperature
preparations revealed various molecular forms with a subunit size
dependency on ALDH activity was observed (data not shown).
of 5 4 kd. We confirmed the thermolability of corneal ALDH (19)
The effect of temperature on the intensity of the 88 kd
and found that enzyme activity is harboured in the 88 kd ALDH
band after SDS-PAGE of purified bovine ALDH as measured with
species. Preincubation of ALDH at high temperatures results in
scanning densitometry showed that it parallelled the decrease in
one protein with a molecular weight of 54 kd.
enzymatic activity (fig.4b). Visualization of aldehyde dehydrogenase activity in a
Sephadex gel filtration of corneal ALDH performed by Evces (19) and Abedinia (1) indicated the presence of a dimer
bovine corneal epithelial extract on the SDS-PAGE gel after the
molecule. This so-called dimer may be identical to the 88 kd
elution of SDS with Triton X-100 revealed that the activity of
protein reported in the present study. The apparent molecular
corneal aldehyde dehydrogenase was associated only with the 88
weight is lower than would be expected. On the other hand
kd band (fig.5). Enzyme activity in the zymograms was not
dimerisation could lead t o conformational changes resulting in an
affected by prior reduction of the samples with DTT. SDS-PAGE
apparent smaller complex than expected in SDS-PAGE analysis.
zymograms of 7 individual human corneal epithelial extracts also
This is the first report showing that the activity of corneal
revealed that the enzyme activity was always present in the 88
ALDH on an SDS-PAGE gel using zymography technique is
kd band (data not shown).
associated with the 88 kd species. This report also confirms the
Current Eye Research observation that the heat lability of corneal ALDH is correlated with a shift of the 88 kd species to the 54 kd form. ACKNOWLEDGEMENTS T.D.G gratefully acknowledge the Hoornvlies Stichting Nederlands and the Nelly Reef Fund for their scholarship.
CORRESPONDING AUTHOR Prof.Dr. A. Kijlstra, Dept. Ophthalmo-Immunology, The Netherlands Ophthalmic Research Institute. P.0.Box 12141
Downloaded by [Imperial College London Library] at 15:38 02 April 2016
1100 A.C. Amsterdam Z.O.The Netherlands.
REFERENCES 1. Abedinia, M., Pain, T., Algar, E.M. and Holmes, R.S.(1990) Bovine corneal aldehyde dehydrogenase: the major soluble corneal protein with a possible dual protective role for the eye. Exp. Eye Res. 419-426. 2. Holmes, R.S. and VandeBerg, J.L.(1986) Ocular NAD-dependent Alcohol dehydrogenase and Aldehyde 383-396. dehydrogenase in the baboon. Exp. Eye Res. 3. Holmes, R.S.(1988) Alcohol dehydrogenases and aldehyde dehydrogenases of anterior eye tissues from humans and other mammals. In "Biomedical and Social Aspects of Alcohol and alcoholism" (eds. Kuriyama, K., Takada, A., and Ishii, H.). Pp. 51-57, Elsevier Science Publisher, Amsterdam, The Netherlands. 4. Alexander, R.J., Silverman, B. and Henley, W.L.(1981) Isolation and characterization of BCP 54, the major soluble protein of bovine cornea. Exp.Eye Res. 2 205-216. 5. Silverman, B., Alexander, R.J. and Henley, W.L.(1981) Tissue and species specificity of BCP 54, the major soluble 19-29. protein of bovine cornea. Exp. Eye Res. 6. Kruit, P.J., Broersma, L., v.d.Gaag, R. and Kijlstra, A.( 1986) Clinical and experimental studies concerning circulating antibodies t o corneal epithelium antigens. Doc. Ophthalmol. & 43-51. 7. Kong, A.S., Henley, W.L. and Luntz, M.H. (1988) lntracellular distribution of corneal protein BCP 5 4 in epithelial cells. In "World Uveitis Symposium" (Eds. Belfort Jr, R., Petrilli A.M.N and Nussenblatt, R.), Pp. 109-1 16. Proceeding of the first world uveitis symposium held in Guaruja' Sao Paulo, Brazil. 8 . Cooper, D.L., Baptist, E.W., Lee, H., Isola, N. and Klintworth, G. K. (1990) Partial aminoacid sequence determination of Bovine Corneal Protein 54 K (BCP 54). Curr. Eye Res. 9.781 -786. 9. Verjans, G.M.G.M., Verhagen, C., Hoeckzema, R. and Kijlstra, A.(1990) Bovine corneal protein 54 (BCP 54) shows strong homology to rat tumor-associated aldehyde dehydrogenase (T-ALDH). Letter to the Editor. Curr. Eye Res. 3, 1217-1218. 10. Cooper, D.L. (1990) Response t o the Editors. Curr. Eye Res. 3, 1219-1220. 11. Cooper, D.L., Baptist, E.W., Enghild, J.J., Isola, N.R. and Klinworth, G.K. (1991 1 Bovine corneal protein 54K (BCP54) is a homologue of the tumor associated(c1ass 3) rat aldehyde dehydrogenase (RATALD).Gene, 98, 201 -207. 12. Verhagen, C.,Hoekzema, R., Verjans, G.M.G.M. and Kijlstra, A. (19911 Identification of bovine corneal protein 5 4 (BCP 54) as an aldehyde dehydrogenase. Letter to the Editor. Exp. 283-284. Eye Res.
a
a,
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