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Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme
Abstract:
Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar.
Insights
Human liver alkaline phosphatase (AP) is a highly glycosylated enzyme composed of two subunits. Its amino-terminal sequence shows significant homology with bovine liver AP and human placental AP isozymes.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Alkaline phosphatase (AP) is a crucial enzyme found in various human tissues.
- Understanding the structural and sequence characteristics of human liver AP is essential for its functional and evolutionary studies.
Purpose of the Study:
- To purify and characterize human liver alkaline phosphatase (AP).
- To determine the molecular weight, subunit composition, and N-terminal amino acid sequence of human liver AP.
- To compare the N-terminal sequences with other AP forms.
Main Methods:
- Homogeneous purification of human liver alkaline phosphatase.
- Molecular weight determination using SDS-PAGE.
- Endoglycosidase F treatment to assess glycosylation.
- Amino-terminal sequencing via Edman degradation.
Main Results:
- Purified human liver AP has a native molecular weight of 150,000 Da, composed of two 75,000 Da subunits.
- Endoglycosidase F treatment reduced the molecular weight to 50,000 Da, indicating extensive glycosylation.
- The N-terminal 22-residue sequence was determined.
- Identical N-terminal sequences were observed between human and bovine liver AP.
- Significant homology was found between the N-termini of human liver and human placental AP isozymes.
Conclusions:
- Human liver AP is a heavily glycosylated dimeric protein.
- The N-terminal sequence is highly conserved between human and bovine liver AP.
- The N-terminal region of human liver AP shares homology with its placental counterpart, suggesting conserved structural or functional roles.