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Purification and cDNA sequence of an inducible nitric oxide synthase from a human tumor cell line
P A Sherman1, V E Laubach, B R Reep
1Division of Experimental Therapy, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Abstract:
A combination of cytokines induced the expression of nitric oxide synthase (NOS) in a human colorectal adenocarcinoma cell line, DLD-1. We have purified the enzyme and examined some of its biochemical properties. An antiserum to an inducible NOS from murine macrophages cross-reacted with the DLD-1 NOS. The purified human and murine enzymes displayed a similar lack of dependence on exogenous calcium and calmodulin for activity, which contrasts with the requirement for calcium and calmodulin of purified brain and endothelial isoforms of NOS. We have also isolated a cDNA for a cytokine-induced NOS from DLD-1 cells. Sequence analysis of this cDNA and NOS cDNAs from human liver, smooth muscle, and macrophages suggests that, at the genetic level, there is a single isoform of human-inducible NOS.
Insights
Cytokines trigger nitric oxide synthase (NOS) in colon cancer cells. This inducible NOS enzyme differs biochemically from brain and endothelial NOS, suggesting a single human inducible NOS gene.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Nitric oxide synthase (NOS) plays a crucial role in various physiological processes.
- Cytokines are known to induce NOS expression in different cell types.
- Understanding the properties of inducible NOS is important for its potential therapeutic targeting.
Purpose of the Study:
- To purify and characterize the cytokine-induced nitric oxide synthase (NOS) from a human colorectal adenocarcinoma cell line (DLD-1).
- To compare the biochemical properties of the DLD-1 NOS with other known NOS isoforms.
- To investigate the genetic basis of human inducible NOS.
Main Methods:
- Purification of nitric oxide synthase (NOS) from DLD-1 cells.
- Biochemical assays to determine enzyme properties, including calcium and calmodulin dependence.
- Western blot analysis using antiserum against murine inducible NOS.
- Isolation and sequencing of a cDNA for cytokine-induced NOS from DLD-1 cells.
- Sequence analysis of NOS cDNAs from various human tissues.
Main Results:
- A combination of cytokines successfully induced NOS expression in DLD-1 cells.
- The purified DLD-1 NOS enzyme was biochemically characterized.
- The DLD-1 NOS showed cross-reactivity with an antiserum to murine inducible NOS.
- The human inducible NOS purified from DLD-1 cells, similar to murine inducible NOS, did not require exogenous calcium and calmodulin for activity.
- This contrasts with the calcium/calmodulin dependence of brain and endothelial NOS isoforms.
- A cDNA for cytokine-induced NOS was isolated from DLD-1 cells.
- Sequence analysis of this cDNA and other human NOS cDNAs indicated a single genetic isoform for human inducible NOS.
Conclusions:
- Cytokine-induced NOS in DLD-1 cells represents a distinct isoform with unique biochemical properties.
- The inducible NOS isoform is independent of calcium and calmodulin, unlike other known NOS types.
- Genetic evidence supports the existence of a single human inducible NOS isoform, despite its expression in various cell types.