ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 297, No. 1, August 15, pp. 80-85, 1992
Purification and Properties of a Protamine Kinase from Bovine Kidney Microsomes’ Grayson
D. Amick,
Department
of Biological
Shrikanth
A. G. Reddy, and Zahi Damuni’
Sciences, CLS 601, The University
of South Carolina,
Columbia, South Carolina 29208
Received March 12, 1992, and in revised form April 20, 1992
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, is: 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-ZOO. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5,8, and ~0.1% activity with casein, histone H2B, and histone Hl, 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 2Az, 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. 0 1992
Academic
Press,
Inc.
The regulation of enzyme activity by reversible phosphorylation is an important mechanism for the coordinated control of cellular activity in response to extracel1 This work was supported in part by Grant DMB-9019882 National Science Foundation. ’ To whom correspondence should be addressed.
from the
lular stimuli (l-5). Over the last few years, it has become evident that cells contain numerous protein kinases (l5). One of these enzymes, designated protamine kinase because of its preference for protamine as substrate, was purified to apparent homogeneity from extracts of bovine kidney cytosol (6). The purified enzyme consisted of a single polypeptide of apparent M, w 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200 (6). Based on its unique catalytic and chromatographic properties, the cytosolic protamine kinase was differentiated from other protein kinases (5, 6), including two forms of a mitochondrial protamine kinase (7). During the course of studies on cytosolic protamine kinase, relatively low levels of protamine kinase activity were detected in a microsomal fraction obtained after precipitation of extracts from bovine kidney with polyethylene glycol (6). Following extraction of this microsomal fraction with 1% Triton X-100, about an eightfold increase in protamine kinase activity was detected. In this paper, we describe the purification and characterization of this microsomal protamine kinase. Based on the substrate specificities, regulatory properties, and peptide patterns obtained following digestion with Staphylococcus aureus V8 proteinase, we conclude that microsomes contain a form of cytosolic protamine kinase. EXPERIMENTAL
PROCEDURES
Materials. S. aureus V8 proteinase was purchased from Sigma. Bovine kidney cytosolic protamine kinase (6) and protein phosphatase 2A2 (8) were purified as described. Purified 40 S ribosomes were a gift from Dr. Gizela Kramer (University of Texas at Austin). The synthetic peptide RRLSSLRA was from Dr. Ruthanan Massarachia (University of Texas at Denton). Other materials are given in Refs. (6, 9, 10). Protein base a&&y. Protamine kinase activity was determined as described (6). One unit of protamine kinase activity was defined as the amount of enzyme that incorporated 1 nmol of 32P into protamine per minute. To ensure linearity, the extent of incorporation of phosphoryl groups was limited to ~3 nmol. To terminate the reactions when the activity of purified preparations of the protamine kinase was determined
80 All
0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
BOVINE
MICROSOMAL
with synthetic peptides as substrates, a 45.~1 aliquot of the reaction mixtures was spotted onto P81 paper and immersed immediately in 30% acetic acid. The paper was then washed extensively with 30% acetic acid, dried, immersed in 10 ml of scintillant, and counted. The activities of pyruvate dehydrogenase phosphatase (8) glucose 6phosphatase (ll), and alkaline phosphodiesterase (12) were determined as described. Protein was determined according to the procedure of Bradford (13). One-dimensional polyacrylamide gel electrophoresis was performed in slab gels as described (14). Two-dimensional gel analysis was performed as described (15). Protein bands were detected by staining with silver (16) or Coomassie blue. Radioactive bands were located with Kodak X-OMAT AR-5 film. N-terminal protein sequence analysis was by the Edman method using an Applied Biosystems Model 477A pulsed liquid phase automated protein sequencer fitted with PTH3 analyzer. Large-scale preparation of microsomes. Highly purified bovine kidney microsomes were prepared essentially as described (17) with some modifications. All operations were performed at 2-5°C. Fresh bovine kidney (1.5 kg) was obtained from a local abattoir and transferred to the laboratory on ice. The kidney cortex was removed and homogenized at high setting in a Waring blender for 1 min in 2 vol of buffer A (25 mM Trischloride, pH 7.3, containing 10% glycerol, 1 mM EDTA, 1 mM benzamfluoride, and 14 mM P-mercaptoidine, 0.1 mM phenylmethylsulfonyl ethanol). The homogenate was centrifuged at 10,000 rpm for 30 min in a Beckman JA-10 rotor. The pH of the solution was adjusted to 7.3 with NaOH. NaCl to a final concentration of 1.0 M was then added with stirring and the pH was readjusted. To this solution was then added with stirring 0.08 vol of 50% (w/v) aqueous poly(ethylene glycol) 8000. After 20 min, the mixture was centrifuged and the precipitate was resuspended in 600 ml of buffer A containing 1.0 M NaCI. To this suspension was then added with stirring 0.08 vol of 50% (w/v) aqueous poly(ethylene) glycol. The solution was centrifuged at 14,000 rpm in a Beckman JA-14 rotor and the supernatant was discarded. The precipitate was resuspended and washed four times as described above with buffer A containing 1.0 M NaCl. The final precipitate was resuspended in buffer A and centrifuged at 14,000 rpm for 30 min in a Beckman JA-14 rotor. The pellets were discarded and the supernatant (microsomal suspension) was used to purify the protamine kinase described in this paper. The microsomal preparations were highly purified as indicated by the specific activity of 1.23 pmol/min/mg obtained for glucose 6-phosphatase. This specific activity is similar to that reported previously for highly purified microsomes (17). The microsomal preparations were essentially free of mitochondrial contamination because little or no pyruvate dehydrogenase phosphatase activity was detected. In addition, relative to homogenates, the microsomes exhibited
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 297, No. 1, August 15, pp. 80-85, 1992
Purification and Properties of a Protamine Kinase from Bovine Kidney Microsomes’ Grayson
D. Amick,
Department
of Biological
Shrikanth
A. G. Reddy, and Zahi Damuni’
Sciences, CLS 601, The University
of South Carolina,
Columbia, South Carolina 29208
Received March 12, 1992, and in revised form April 20, 1992
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, is: 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-ZOO. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5,8, and ~0.1% activity with casein, histone H2B, and histone Hl, 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 2Az, 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. 0 1992
Academic
Press,
Inc.
The regulation of enzyme activity by reversible phosphorylation is an important mechanism for the coordinated control of cellular activity in response to extracel1 This work was supported in part by Grant DMB-9019882 National Science Foundation. ’ To whom correspondence should be addressed.
from the
lular stimuli (l-5). Over the last few years, it has become evident that cells contain numerous protein kinases (l5). One of these enzymes, designated protamine kinase because of its preference for protamine as substrate, was purified to apparent homogeneity from extracts of bovine kidney cytosol (6). The purified enzyme consisted of a single polypeptide of apparent M, w 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200 (6). Based on its unique catalytic and chromatographic properties, the cytosolic protamine kinase was differentiated from other protein kinases (5, 6), including two forms of a mitochondrial protamine kinase (7). During the course of studies on cytosolic protamine kinase, relatively low levels of protamine kinase activity were detected in a microsomal fraction obtained after precipitation of extracts from bovine kidney with polyethylene glycol (6). Following extraction of this microsomal fraction with 1% Triton X-100, about an eightfold increase in protamine kinase activity was detected. In this paper, we describe the purification and characterization of this microsomal protamine kinase. Based on the substrate specificities, regulatory properties, and peptide patterns obtained following digestion with Staphylococcus aureus V8 proteinase, we conclude that microsomes contain a form of cytosolic protamine kinase. EXPERIMENTAL
PROCEDURES
Materials. S. aureus V8 proteinase was purchased from Sigma. Bovine kidney cytosolic protamine kinase (6) and protein phosphatase 2A2 (8) were purified as described. Purified 40 S ribosomes were a gift from Dr. Gizela Kramer (University of Texas at Austin). The synthetic peptide RRLSSLRA was from Dr. Ruthanan Massarachia (University of Texas at Denton). Other materials are given in Refs. (6, 9, 10). Protein base a&&y. Protamine kinase activity was determined as described (6). One unit of protamine kinase activity was defined as the amount of enzyme that incorporated 1 nmol of 32P into protamine per minute. To ensure linearity, the extent of incorporation of phosphoryl groups was limited to ~3 nmol. To terminate the reactions when the activity of purified preparations of the protamine kinase was determined
80 All
0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
BOVINE
MICROSOMAL
with synthetic peptides as substrates, a 45.~1 aliquot of the reaction mixtures was spotted onto P81 paper and immersed immediately in 30% acetic acid. The paper was then washed extensively with 30% acetic acid, dried, immersed in 10 ml of scintillant, and counted. The activities of pyruvate dehydrogenase phosphatase (8) glucose 6phosphatase (ll), and alkaline phosphodiesterase (12) were determined as described. Protein was determined according to the procedure of Bradford (13). One-dimensional polyacrylamide gel electrophoresis was performed in slab gels as described (14). Two-dimensional gel analysis was performed as described (15). Protein bands were detected by staining with silver (16) or Coomassie blue. Radioactive bands were located with Kodak X-OMAT AR-5 film. N-terminal protein sequence analysis was by the Edman method using an Applied Biosystems Model 477A pulsed liquid phase automated protein sequencer fitted with PTH3 analyzer. Large-scale preparation of microsomes. Highly purified bovine kidney microsomes were prepared essentially as described (17) with some modifications. All operations were performed at 2-5°C. Fresh bovine kidney (1.5 kg) was obtained from a local abattoir and transferred to the laboratory on ice. The kidney cortex was removed and homogenized at high setting in a Waring blender for 1 min in 2 vol of buffer A (25 mM Trischloride, pH 7.3, containing 10% glycerol, 1 mM EDTA, 1 mM benzamfluoride, and 14 mM P-mercaptoidine, 0.1 mM phenylmethylsulfonyl ethanol). The homogenate was centrifuged at 10,000 rpm for 30 min in a Beckman JA-10 rotor. The pH of the solution was adjusted to 7.3 with NaOH. NaCl to a final concentration of 1.0 M was then added with stirring and the pH was readjusted. To this solution was then added with stirring 0.08 vol of 50% (w/v) aqueous poly(ethylene glycol) 8000. After 20 min, the mixture was centrifuged and the precipitate was resuspended in 600 ml of buffer A containing 1.0 M NaCI. To this suspension was then added with stirring 0.08 vol of 50% (w/v) aqueous poly(ethylene) glycol. The solution was centrifuged at 14,000 rpm in a Beckman JA-14 rotor and the supernatant was discarded. The precipitate was resuspended and washed four times as described above with buffer A containing 1.0 M NaCl. The final precipitate was resuspended in buffer A and centrifuged at 14,000 rpm for 30 min in a Beckman JA-14 rotor. The pellets were discarded and the supernatant (microsomal suspension) was used to purify the protamine kinase described in this paper. The microsomal preparations were highly purified as indicated by the specific activity of 1.23 pmol/min/mg obtained for glucose 6-phosphatase. This specific activity is similar to that reported previously for highly purified microsomes (17). The microsomal preparations were essentially free of mitochondrial contamination because little or no pyruvate dehydrogenase phosphatase activity was detected. In addition, relative to homogenates, the microsomes exhibited
