Serine-palmitoyl transferase activity in cultured human keratinocytes
Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We re...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
1990-09-01
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| Series: | Journal of Lipid Research |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520423491 |
| _version_ | 1818927443647397888 |
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| author | WM Holleran ML Williams WN Gao PM Elias |
| author_facet | WM Holleran ML Williams WN Gao PM Elias |
| author_sort | WM Holleran |
| collection | DOAJ |
| description | Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We report here the initial characterization of serine-palmitoyl transferase (EC 2.3.1.50; SPT), the rate-limiting enzyme in the synthesis of sphingolipids, from cultured human neonatal keratinocytes. Subcellular fractionation studies demonstrated that 79% of the total cellular SPT activity was associated with the microsomes. The specific activity of keratinocyte SPT was 270 +/- 20 pmol/min per mg of microsomal protein, a level significantly higher than activities reported in other tissues. Keratinocyte SPT showed an apparent Km for L-serine of 0.40 (+/- 0.04 mM, with an alkaline pH optimum (8.2 +/- 0.4). Keratinocyte SPT utilizes palmitoyl-CoA preferentially over other saturated or unsaturated acyl-CoA substrates; increasing acyl-CoA chain lengths above C16 by one or two carbons was less detrimental to activity than similar decrements in chain length. Finally, the mechanism-based inhibitors L-cycloserine and beta-chloro-L-alanine, demonstrated potent inhibition of keratinocyte SPT activity, with 50% inhibitory concentrations of approximately 3.0 and 25 microM, respectively. In summary, we have found that cultured human neonatal keratinocytes contain unusually high levels of serine-palmitoyl transferase activity, and that the substrate specificity of keratinocyte SPT may determine the base composition of epidermal sphingolipids. |
| first_indexed | 2024-12-20T03:13:06Z |
| format | Article |
| id | doaj.art-22cfa0a326d1469fa2639a9b6e79e84d |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-20T03:13:06Z |
| publishDate | 1990-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-22cfa0a326d1469fa2639a9b6e79e84d2022-12-21T19:55:25ZengElsevierJournal of Lipid Research0022-22751990-09-0131916551661Serine-palmitoyl transferase activity in cultured human keratinocytesWM Holleran0ML Williams1WN Gao2PM Elias3Dermatology Service, Veterans Administration Medical Center, San Francisco, CA.Dermatology Service, Veterans Administration Medical Center, San Francisco, CA.Dermatology Service, Veterans Administration Medical Center, San Francisco, CA.Dermatology Service, Veterans Administration Medical Center, San Francisco, CA.Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We report here the initial characterization of serine-palmitoyl transferase (EC 2.3.1.50; SPT), the rate-limiting enzyme in the synthesis of sphingolipids, from cultured human neonatal keratinocytes. Subcellular fractionation studies demonstrated that 79% of the total cellular SPT activity was associated with the microsomes. The specific activity of keratinocyte SPT was 270 +/- 20 pmol/min per mg of microsomal protein, a level significantly higher than activities reported in other tissues. Keratinocyte SPT showed an apparent Km for L-serine of 0.40 (+/- 0.04 mM, with an alkaline pH optimum (8.2 +/- 0.4). Keratinocyte SPT utilizes palmitoyl-CoA preferentially over other saturated or unsaturated acyl-CoA substrates; increasing acyl-CoA chain lengths above C16 by one or two carbons was less detrimental to activity than similar decrements in chain length. Finally, the mechanism-based inhibitors L-cycloserine and beta-chloro-L-alanine, demonstrated potent inhibition of keratinocyte SPT activity, with 50% inhibitory concentrations of approximately 3.0 and 25 microM, respectively. In summary, we have found that cultured human neonatal keratinocytes contain unusually high levels of serine-palmitoyl transferase activity, and that the substrate specificity of keratinocyte SPT may determine the base composition of epidermal sphingolipids.http://www.sciencedirect.com/science/article/pii/S0022227520423491 |
| spellingShingle | WM Holleran ML Williams WN Gao PM Elias Serine-palmitoyl transferase activity in cultured human keratinocytes Journal of Lipid Research |
| title | Serine-palmitoyl transferase activity in cultured human keratinocytes |
| title_full | Serine-palmitoyl transferase activity in cultured human keratinocytes |
| title_fullStr | Serine-palmitoyl transferase activity in cultured human keratinocytes |
| title_full_unstemmed | Serine-palmitoyl transferase activity in cultured human keratinocytes |
| title_short | Serine-palmitoyl transferase activity in cultured human keratinocytes |
| title_sort | serine palmitoyl transferase activity in cultured human keratinocytes |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520423491 |
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