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Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine
Abstract:
Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg(2+) and ATP, but at low concentrations of Mg(2+) and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca(2+) and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg(2+) and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues.
Insights
Cyclic AMP significantly enhances protein phosphorylation in bovine corpus luteum smooth microsomes, unlike free ribosomes. This cyclic AMP-dependent activity is similar to soluble protein kinase and involves phosphoserine.
Area of Science:
- Biochemistry
- Cell Biology
- Reproductive Biology
Background:
- Protein phosphorylation is a key regulatory mechanism in cellular processes.
- Cyclic AMP (cAMP) acts as a crucial second messenger in various signaling pathways.
- The corpus luteum plays a vital role in reproductive function.
Purpose of the Study:
- To investigate the self-phosphorylation activity of free ribosomes and smooth microsomes from bovine corpus luteum.
- To determine the role of cyclic AMP (cAMP) in modulating this phosphorylation.
- To characterize the properties of cAMP-dependent and independent phosphorylation in these fractions.
Main Methods:
- Preparation of free ribosomes and smooth microsomes from bovine corpus luteum.
- In vitro self-phosphorylation assays using Mg(2+) and ATP.
- Assessment of cAMP and cyclic GMP (cGMP) effects on phosphorylation.
- Analysis of phosphorylated residues (phosphoserine, phosphothreonine) and protein identification via SDS-PAGE.
Main Results:
- Both ribosome and microsomal fractions exhibit self-phosphorylation.
- Smooth microsomes show significantly higher cAMP-dependent phosphorylation (up to 10-fold stimulation) compared to free ribosomes, especially at low Mg(2+) and ATP concentrations.
- cAMP-dependent phosphorylation is inhibited by Ca(2+) and a heat-stable inhibitor protein, similar to soluble cAMP-dependent protein kinase.
- cGMP can activate the reaction at higher concentrations than cAMP.
- Phosphorylated proteins are identified, with phosphoserine being the predominant residue in cAMP-stimulated reactions.
- A distinct cAMP-independent phosphorylation occurs at high Mg(2+) and ATP concentrations, resistant to the inhibitor protein, and involves both threonine and serine.
Conclusions:
- Cyclic AMP plays a significant role in regulating protein phosphorylation in bovine corpus luteum smooth microsomes.
- The smooth microsomal fraction contains a cAMP-dependent protein kinase activity distinct from free ribosomes.
- These findings highlight the importance of cAMP-mediated signaling in the functional regulation of the corpus luteum.