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Multiple polypeptides immunologically related to beta-poly(L-malate) hydrolase (polymalatase) in the plasmodium of
1Institut für Biophysik und physikalische Biochemie der Universität, Regensburg, Germany.
Abstract:
Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.
Insights
Physarum polycephalum contains beta-poly(L-malate) and polymalatase. This enzyme is derived from a precursor protein, suggesting a role beyond hydrolase activity, possibly in beta-poly(L-malate) transport.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- The slime mold Physarum polycephalum synthesizes beta-poly(L-malate), a specific polyanion.
- A corresponding hydrolase, polymalatase, is also expressed in the plasmodial stage.
Purpose of the Study:
- To investigate the nature and function of polymalatase in Physarum polycephalum.
- To determine the origin and potential roles of polymalatase and its associated proteins.
Main Methods:
- Partial purification of polymalatase.
- Identification of polymalatase polypeptides (68 kDa and 97 kDa) using SDS-PAGE.
- Deglycosylation of polymalatase forms.
- Antibody-based detection of intracellular protein distribution.
- Two-dimensional gel electrophoresis (non-denaturing/SDS-denaturing).
Main Results:
- Polymalatase preparation contained multiple polypeptides, including 68 kDa and 97 kDa forms, both glycosylated.
- Deglycosylation yielded 54 kDa and 88 kDa forms, respectively.
- Antisera cross-reacted with multiple polypeptides ubiquitously distributed in the plasmodium.
- Proteins appeared to be derived from a 200 kDa precursor via proteolytic fragmentation.
- Polymalatase activity could be generated from a high molecular-mass precursor within the plasmodium.
Conclusions:
- Polymalatase is likely generated from a large precursor protein through proteolytic cleavage within the plasmodium.
- The ubiquitous distribution and intracellular conditions suggest polymalatase may have functions beyond simple hydrolysis, potentially involving beta-poly(L-malate) transport.