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Purification and properties of a protamine kinase from bovine kidney microsomes
G D Amick1, S A Reddy, Z Damuni
1Department of Biological Sciences, University of South Carolina, Columbia 29208.
Abstract:
About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.
Insights
A protamine kinase was identified in bovine kidney microsomes, showing significant activity and phosphorylating ribosomal protein S6. This microsomal enzyme shares properties with its cytosolic counterpart, indicating its presence in both cellular fractions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Microsomes, derived from endoplasmic reticulum, are crucial cellular components involved in protein synthesis and modification.
- Protamine kinase activity has been previously identified in the cytosol, but its presence and function within microsomal fractions remained unclear.
Purpose of the Study:
- To investigate the presence and characteristics of protamine kinase activity within bovine kidney microsomes.
- To purify and characterize the microsomal protamine kinase and compare it with the known cytosolic enzyme.
Main Methods:
- Microsomal extraction using Triton X-100.
- Enzyme purification via chromatography.
- Protein characterization using SDS-PAGE and gel permeation chromatography.
- Substrate specificity assays using protamine, synthetic peptides, and histones.
- Identification of phosphorylated substrates using N-terminal sequencing.
- Comparison with cytosolic protamine kinase using peptide mapping.
Main Results:
- Protamine kinase activity was significantly enriched in purified bovine kidney microsomes.
- The microsomal protamine kinase was purified to homogeneity, with an estimated molecular mass of 45,000 Da.
- The purified kinase showed high activity towards protamine and a synthetic peptide, with minimal activity against casein and histones.
- The enzyme phosphorylated several 40S ribosome polypeptides, including ribosomal protein S6.
- The microsomal protamine kinase exhibited identical properties and peptide patterns to the previously characterized cytosolic protamine kinase.
Conclusions:
- A protamine kinase, biochemically similar to the cytosolic form, is present in bovine kidney microsomes.
- This microsomal protamine kinase phosphorylates ribosomal protein S6, suggesting a role in ribosome biogenesis or function.
- The findings indicate that the cytosolic protamine kinase may also exist in a microsomal-associated form.