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Influence of carbohydrate side chains on activity of tissue-type plasminogen activator
Abstract:
When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.
Insights
Messenger RNA translation yields a 52 kDa tissue-type plasminogen activator without activity. Adding microsomes produces an active 63 kDa form, suggesting glycosylation impacts activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Messenger RNA (mRNA) translation in reticulocyte lysates is a common method to study protein synthesis.
- Tissue-type plasminogen activator (t-PA) is a key enzyme in fibrinolysis.
- Post-translational modifications, such as glycosylation, can significantly alter protein function.
Purpose of the Study:
- To investigate the role of post-translational modifications in the activation of tissue-type plasminogen activator (t-PA).
- To determine how glycosylation affects the biological activity and molecular weight of t-PA.
Main Methods:
- In vitro translation of mRNA from melanoma cells using reticulocyte lysates.
- Supplementation of translation systems with microsomal membranes.
- Immunoprecipitation of t-PA using specific antibodies.
- Enzymatic treatment of natural t-PA with glycosidases (neuraminidase, beta-galactosidase, alpha-mannosidase).
- Analysis of protein molecular weight and biological activity.
Main Results:
- Translation of mRNA produced a 52 kDa t-PA precursor lacking biological activity.
- Supplementation with microsomes resulted in a 63 kDa active t-PA form.
- Glycosidase treatment altered t-PA electrophoretic mobility and biological activity; neuraminidase and beta-galactosidase decreased activity, while alpha-mannosidase increased it.
Conclusions:
- The carbohydrate moiety of t-PA plays a crucial role in its biological activity.
- Glycosylation is essential for the proper maturation and activation of t-PA.
- These findings highlight the importance of post-translational modifications in protein function.