Related Experiment Videos
Isolation of soluble tyrosinase from human melanoma cells
A Wittbjer1, G Odh, A M Rosengren
1Department of Pharmacology, University of Lund, Sweden.
Abstract:
In the human melanoma cell tyrosinase exists in a membraneous and a soluble form. The membraneous enzyme has an N-terminal amino acid sequence identical to that predicted from a human c-DNA clone by Kwon et al.. The soluble form has now been isolated by a technique mainly based on the trypsin resistence of the enzyme and the use of hydrophobic interaction chromatography. The specific dopa oxidase activity of the soluble enzyme was 300 mumol/min x mg protein. On isoelectric focusing the enzyme was found in at least ten bands, pI between 3.8-4.6. The molecular weight was found to be 53,000 D. The N-terminal amino acid sequence was the same as that found in the membrane bound form of the enzyme, i.e. the protein maps at the c-albino locus.
Insights
Researchers isolated a soluble form of human melanoma cell tyrosinase using trypsin resistance and chromatography. This soluble enzyme shares an N-terminal sequence with the membrane-bound form, mapping to the c-albino locus.
Area of Science:
- Biochemistry
- Molecular Biology
- Melanoma Research
Background:
- Tyrosinase is a key enzyme in melanin production, existing in both membrane-bound and soluble forms within human melanoma cells.
- Previous studies identified the N-terminal sequence of membrane-bound tyrosinase from a human cDNA clone.
- Understanding the characteristics of soluble tyrosinase is crucial for melanoma research.
Purpose of the Study:
- To isolate and characterize the soluble form of human melanoma cell tyrosinase.
- To compare the N-terminal amino acid sequence of soluble tyrosinase with its membrane-bound counterpart.
- To investigate the enzymatic activity and physical properties of soluble tyrosinase.
Main Methods:
- Enzyme isolation utilizing trypsin resistance and hydrophobic interaction chromatography.
- Measurement of specific dopa oxidase activity.
- Isoelectric focusing to determine the enzyme's charge heterogeneity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weight.
Main Results:
- The soluble form of tyrosinase was successfully isolated from human melanoma cells.
- The enzyme exhibited a specific dopa oxidase activity of 300 μmol/min x mg protein.
- Isoelectric focusing revealed at least ten bands with pI values between 3.8-4.6, indicating charge heterogeneity.
- The molecular weight of the soluble enzyme was determined to be 53,000 D.
- The N-terminal amino acid sequence of soluble tyrosinase was identical to the membrane-bound form, consistent with the c-albino locus.
Conclusions:
- Soluble tyrosinase from human melanoma cells shares identical N-terminal sequences with the membrane-bound form.
- The characterization of soluble tyrosinase provides insights into its role in melanoma.
- Further research can explore the functional differences and regulatory mechanisms of tyrosinase isoforms in melanoma progression.