Andrew Paterson

Kinetic Analysis of Phospholipase C Isoforms Using Phospholipid-Detergent Mixed Micelles EVIDENCE FOR INTERFACIAL CATALYSIS INVOLVING DISTINCT MICELLE BINDING AND CATALYTIC STEPS

Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) hydrolysis by three different β-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regulated enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4, 5) P2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essentially inert diluent of PtdIns (4, 5) P2 with respect to PLCβ activity. Kinetic analyses were performed to test whether PLCβ activity was dependent on both bulk PtdIns (4, 5) P2 concentration and surface concentration in the micelles as has been shown for other lipid metabolising enzymes. Each of the PLCβ isoforms behaved similarly in these analyses, which indicated the involvement of at least two binding events. Interfacial Michaelis constants were calculated to be between 0.1-0.2 mol fraction for all three enzymes, and K(the equilibrium dissociation constant of PLC for lipid) ranged between 100-200 μM. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLCβ oligomerization implying that PLCβ monomers possess more than one lipid-binding site. Surface dilution of PLC-catalyzed PtdIns (4, 5) P2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, which profoundly reduced PLC activity, suggesting that these lipids may inhibit enzyme action. The data indicate that G protein-regulated isoforms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLCβ isoforms operate under first-order conditions. These findings may have implications for the mechanisms of regulation of PLCβs by G protein subunits.

Concentration of enzyme-dependent activation of PLC-β1 and PLC-β2 by Gα11 and βγ-subunits

Abstract Differential regulation of PLC-β1 and −β2 by the G-protein α-subunit, Gα11, and by G-protein βγ-subunits was studied utilizing recombinant PLC-β1 and −β2. Rat PLC-β1 and human PLC-β2 were purified after recombinant baculovirus-mediated expression in Sf9 cells. The catalytic properties of the purified recombinant isoenzymes were directly compared to PLC-β1 purified from bovine brain and PLC-β2 partially purified from HL60 polymorphonuclear neutrophils. The recombinant isoenzymes were indistinguishable from the native isoenzymes with respect to dependence of reaction velocity on bulk PtdIns(4,5)P2 substrate concentration, pH, and free Ca2+ concentration. Marked AlF4t--dependent activation was observed upon reconstitution of rPLC-β1 with the G-protein α-subunit, Gα11. Activation occurred with a concentration dependence on Gα11 for activation and elevation in reaction velocity that was similar to that of native PLC-β1. In contrast, Gα11 promoted only a small elevation in the catalytic rate of recombinant PLC-β2, which was also typical of the native isoenzyme. Maximal reaction rates with respect to PLC-β isoenzyme concentration were achieved and indicated that rPLC-β2 required 10-fold greater concentrations of both Gα11 and of rPLC-β2 for activation than did rPLC-β1. rPLC-β1 and rPLC-β2 were also differentially regulated by βγ-subunits. This differential activation was not the result of different concentration dependencies on βγ-subunit for activation, but rather, the result of the greater degree to which the catalytic rate of PLC-β2 was elevated by βγ-subunits when compared to PLC-β1.

PURIFICATION AND G PROTEIN SUBUNIT REGULATION OF A PHOSPHOLIPASE C-BETA FROM XENOPUS LAEVIS OOCYTES

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Gαi, Gαo, Gαq, Gαs, and Gβγ all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-βX) that exhibits homology to the PLC-β class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-βX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-βX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-βX was activated by both Gαq and Gβγ. PLC-βX exhibited a higher apparent affinity for Gαq than Gβγ, and Gαq was more efficacious than Gβγ at lower concentrations of PLC-βX. Relative to other PLC-β isoenzymes, PLC-βX was less sensitive to stimulation by Gαq than PLC-β1 but similar to PLC-β2 and PLC-βT. PLC-βX was more sensitive to stimulation by Gβγ than PLC-β1 but less sensitive than PLC-β2 and PLC-βT. In contrast PLC-βX was not activated by the pertussis toxin substrate G proteins Gαi1, Gαi2, Gαi3, or Gαo. These results are consistent with the idea that PLC-βX is regulated by α-subunits of the Gq family and by Gβγ and do not support the idea that α-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.

G-protein-mediated regulation of phospholipase C: Involvement of βγ subunits

The protein components of the G-protein-linked cell surface receptor-regulated inositol lipid-signaling cascade have been identified recently. The G(q) family of G-protein α subunits is responsible for pertussis toxin-insensitive activation of a family of phospholipase C-β isoenzymes. However, it also has been shown that G-protein βγ subunits activate certain of these phospholipase C-β isoenzymes, and that this novel activity may account for activation of phospholipase C by G proteins that are not members of the G(q) family, including those that account for pertussis-toxin-sensitive inositol lipid signaling.

[17] Expression, purification, and functional reconstitution of recombinant phospholipase C-β isozymes

Publisher Summary This chapter is a study on the mechanisms by which Phospholipase C-β isoenzymes that are regulated requires the acquisition of substantial quantities or purified protein. Phospholipase C-β1 and C-β2 can be purified from bovine brain and from cultured polymorphonuclear neutrophils, respectively. In both the cases, the purification procedure is protracted, requiring access to large quantities of starting material and with only moderate or poor yield of purified protein. The chapter describes methodology for the production of recombinant PLC-β1 and-β2 in the Spodopterafrugiperda (Sf9)/baculovirus expression system. This system has the advantage of producing milligram quantities of homogeneous recombinant PLC-β1 and PLC-β2 from relatively small cultures of Sf9 cells. The smaller cultures and higher levels of recombinant protein expression facilitate their isolation by simpler purification protocols. The chapter describes the methodology for the investigation of the mechanism by which α and βγ subunits regulate the activity of the PLC-β isoforms. Utilization of the recombinant baculovirus/Sf9 expression system has allowed production of recombinant PLC-β1 and PLC-β2, both with properties that are indistinct from those of the same isoenzymes purified from native sources.

Regulation of Phospholipase C-β Isoenzymes

A broad range of hormones, neurotransmitters, growth factors, and chemoattractants produce their physiological effects through phospholipase C (PLC)-catalyzed initiation of the inositol lipid signaling cascade (Berridge and Irvine, 1987). Although tyrosine phosphorylation of the SH2- and SH3-domain-containing PLC-γ isoenzymes accounts for interface of a number of growth factors with this signaling pathway, the majority of extracellular stimuli act through seven-transmembrane-spanning receptors that activate G-proteins that in turn activate isoenzymes of the PLC-β family (Rhee and Choi, 1992). The focus of this chapter is on this latter group of signaling proteins.