Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits

J L Blank; K Shaw; A H Ross; J H Exton

doi:10.1016/S0021-9258(19)74586-4

Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits

J L Blank, K Shaw, A H Ross, J H Exton

Vanderbilt University School of Medicine

Research output: Contribution to journal › Article › peer-review

Abstract

We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein beta gamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta 1, 145-kDa PLC-gamma 1, and 85-kDa PLC-delta 1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta 3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta 3. Reconstitution of purified PLC-110 with beta gamma-subunits produced greater than 100-fold activation, indicating activation was observed at approximately 60 nM beta gamma and full activation at approximately 500 nM beta gamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 microM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha q that fully activated PLC-beta 1 failed to stimulate PLC-110. This observation indicates that the site at which beta gamma interacts with PLC-110 is distinct from that at which alpha q regulates the activity of PLC-beta isozymes.

Original language	English
Pages (from-to)	25184-25191
Number of pages	8
Journal	Journal of Biological Chemistry
Volume	268
Issue number	33
DOIs	https://doi.org/10.1016/S0021-9258(19)74586-4
Publication status	Published - 25 Nov 1993
Externally published	Yes

Keywords

Animals
Blotting, Western
Calcium/metabolism
Cattle
Chromatography, Gel
Chromatography, Ion Exchange
Cross Reactions
Enzyme Activation
GTP-Binding Proteins/metabolism
Guanosine 5'-O-(3-Thiotriphosphate)/metabolism
Phosphatidylinositol Diacylglycerol-Lyase
Phosphoric Diester Hydrolases/isolation & purification
Substrate Specificity

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@article{93e4628a05954cd9918ef371540bcf60,

title = "Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits",

abstract = "We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein beta gamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4\%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta 1, 145-kDa PLC-gamma 1, and 85-kDa PLC-delta 1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta 3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta 3. Reconstitution of purified PLC-110 with beta gamma-subunits produced greater than 100-fold activation, indicating activation was observed at approximately 60 nM beta gamma and full activation at approximately 500 nM beta gamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 microM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha q that fully activated PLC-beta 1 failed to stimulate PLC-110. This observation indicates that the site at which beta gamma interacts with PLC-110 is distinct from that at which alpha q regulates the activity of PLC-beta isozymes.",

keywords = "Animals, Blotting, Western, Calcium/metabolism, Cattle, Chromatography, Gel, Chromatography, Ion Exchange, Cross Reactions, Enzyme Activation, GTP-Binding Proteins/metabolism, Guanosine 5'-O-(3-Thiotriphosphate)/metabolism, Phosphatidylinositol Diacylglycerol-Lyase, Phosphoric Diester Hydrolases/isolation \& purification, Substrate Specificity",

author = "Blank, \{J L\} and K Shaw and Ross, \{A H\} and Exton, \{J H\}",

year = "1993",

month = nov,

day = "25",

doi = "10.1016/S0021-9258(19)74586-4",

language = "English",

volume = "268",

pages = "25184--25191",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

number = "33",

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TY - JOUR

T1 - Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits

AU - Blank, J L

AU - Shaw, K

AU - Ross, A H

AU - Exton, J H

PY - 1993/11/25

Y1 - 1993/11/25

N2 - We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein beta gamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta 1, 145-kDa PLC-gamma 1, and 85-kDa PLC-delta 1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta 3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta 3. Reconstitution of purified PLC-110 with beta gamma-subunits produced greater than 100-fold activation, indicating activation was observed at approximately 60 nM beta gamma and full activation at approximately 500 nM beta gamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 microM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha q that fully activated PLC-beta 1 failed to stimulate PLC-110. This observation indicates that the site at which beta gamma interacts with PLC-110 is distinct from that at which alpha q regulates the activity of PLC-beta isozymes.

AB - We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein beta gamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta 1, 145-kDa PLC-gamma 1, and 85-kDa PLC-delta 1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta 3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta 3. Reconstitution of purified PLC-110 with beta gamma-subunits produced greater than 100-fold activation, indicating activation was observed at approximately 60 nM beta gamma and full activation at approximately 500 nM beta gamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 microM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha q that fully activated PLC-beta 1 failed to stimulate PLC-110. This observation indicates that the site at which beta gamma interacts with PLC-110 is distinct from that at which alpha q regulates the activity of PLC-beta isozymes.

KW - Animals

KW - Blotting, Western

KW - Calcium/metabolism

KW - Cattle

KW - Chromatography, Gel

KW - Chromatography, Ion Exchange

KW - Cross Reactions

KW - Enzyme Activation

KW - GTP-Binding Proteins/metabolism

KW - Guanosine 5'-O-(3-Thiotriphosphate)/metabolism

KW - Phosphatidylinositol Diacylglycerol-Lyase

KW - Phosphoric Diester Hydrolases/isolation & purification

KW - Substrate Specificity

U2 - 10.1016/S0021-9258(19)74586-4

DO - 10.1016/S0021-9258(19)74586-4

M3 - Article

C2 - 8227082

SN - 0021-9258

VL - 268

SP - 25184

EP - 25191

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 33

ER -

Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits

Abstract

Keywords

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