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A 138-kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity
R A Greiner1, D Jacobs-Krahnen, R Mutzel
1Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Abstract:
A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide.
Insights
Dictyostelium discoideum membranes contain a folate deaminase glycoprotein with two distinct folate binding sites. One site is catalytic, while the other may be involved in folate perception during chemotaxis.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Dictyostelium discoideum is a model organism for studying cellular processes.
- Folate metabolism and signaling play crucial roles in cellular functions.
- Understanding folate binding and deamination is key to deciphering cellular responses.
Purpose of the Study:
- To purify and characterize the folate deaminase glycoprotein from Dictyostelium discoideum.
- To investigate the binding characteristics and potential functions of different folate binding sites on the enzyme.
- To explore the relationship between folate deamination and chemotactic perception.
Main Methods:
- Purification of a 138-kDa glycoprotein with folate deaminase activity from Dictyostelium discoideum membranes.
- Enzyme inhibition studies using p-chloromercuriphenylsulfonate to protect folate.
- Analysis of folate binding affinities and specificities to the purified glycoprotein.
- Comparison of binding site preferences with observed chemotaxis responses.
Main Results:
- A 138-kDa glycoprotein exhibiting folate deaminase activity was isolated.
- Two distinct folate binding sites were identified on the deaminase: one high-affinity catalytic site and one lower-affinity site.
- The lower-affinity site showed a preference for N10-methylfolate over folate, mirroring observations in chemotaxis assays.
- These findings suggest a dual role for the polypeptide in both folate deamination and chemotactic perception.
Conclusions:
- The folate deaminase glycoprotein from Dictyostelium discoideum possesses multiple functional domains.
- The enzyme's catalytic site and a separate binding site are involved in distinct folate interactions.
- A single polypeptide may mediate both the enzymatic inactivation of folate and its sensory perception in chemotaxis.