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Multiple forms of alkaline phosphatases in human liver tissue
Clinica Chimica Acta; International Journal of Clinical Chemistry
|December 15, 1984
Summary
Human liver alkaline phosphatases (AP) were separated into two distinct forms, APII and APIIII, using affinity chromatography. These forms share similar kinetic properties but differ in molecular size, suggesting modifications to a single enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Alkaline phosphatases (AP) are crucial enzymes involved in various physiological processes.
- Multiple forms of AP exist, differing in tissue distribution and biochemical properties.
- Understanding the structural and functional differences between AP isoforms is essential for comprehending their roles in health and disease.
Purpose of the Study:
- To isolate and characterize distinct forms of alkaline phosphatase from human liver.
- To investigate the biochemical and kinetic properties of purified AP fractions.
- To elucidate the relationship between different liver AP forms and their potential origins.
Main Methods:
- Extraction of alkaline phosphatases from human liver in the presence of n-butanol.
- Separation of AP fractions using affinity chromatography on phenylsepharose Cl-4B.
- Purification of AP fractions (APII and APIIII) via repeated chromatography.
- Enzyme activity assays, including specific activity determination.
- Analysis of chromatographic and electrophoretic behavior.
- Assessment of enzyme kinetic properties (substrate activity, thermolability, inhibitor sensitivity).
Main Results:
- Two distinct AP fractions, APII and APIIII, were successfully separated and purified.
- APII was purified to a single enzyme entity with high specific activity (1,684 kU/g protein).
- APIII was purified with lower specific activity (535 kU/g protein) and retained gamma-glutamyltransferase activity.
- APIII was found to be a larger molecule than APII based on chromatographic and electrophoretic behavior.
- Both APII and APIIII exhibited similar enzyme kinetic characteristics, including substrate activity, thermolability, and inhibitor sensitivity.
Conclusions:
- The purified liver alkaline phosphatase forms (APII and APIIII) possess identical active centers, indicating they are likely modified forms of a single enzyme.
- Differences in chromatographic and electrophoretic behavior are attributed to modifications in protein residues rather than alterations in the active site.
- These liver AP forms are distinct from the AP isoenzyme observed in serum of cholestatic patients.