Application of high-pressure to subfractionate membrane protein-lipid complexes: A case study of protein kinase C
Abstract
Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subrractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical cotact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.
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Cited by (23)
A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability
2016, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :While HP is usually regarded as constant and its influence is ignored in many biochemical experiments, the physicochemical effects of high hydrostatic pressure (HHP) on biomolecules, cells and tissues have been studied [5–7]. For example, HHP has been utilized in the solubilization of membrane-associated protein kinase C without detergent [8], and in the preparation of various proteins, lipids and nucleic acids [9]. In addition to static HHP treatment, dynamic HHP treatments, such as pressure cycling, have been developed and utilized for diverse biochemical approaches [9].
The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF + insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200 MPa) at room temperature for 1–16 h in the presence of 0–2.0% (w/v) n-dodecyl-β-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~ 1.5-fold after 1 h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6 h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.
Agents that act by different mechanisms modulate the activity of protein kinase CβII isozyme in the rat spinal cord during peripheral inflammation
2006, NeuroscienceHyperalgesia following unilateral complete Freund’s adjuvant-induced inflammation was characterized by paw withdrawal latency to thermal stimulus. Paw withdrawal latencies were significantly shorter on the complete Freund’s adjuvant-treated paw than on the contralateral paw of the complete Freund’s adjuvant- and the sham-treated rats. Total cytosolic protein kinase C activity in the lumbar enlargement was unchanged on the sides of the spinal cord ipsi- and contra-lateral to the inflamed paw. Membrane-associated activities of protein kinase Cα, protein kinase CβI and protein kinase Cγ did not change significantly on the sides of the cord ipsi- and contra-lateral to the inflammation. However, membrane-associated activity of protein kinase CβII was increased in the cord section ipsilateral to the inflammation, suggesting that increased translocation/activation of protein kinase CβII is related to thermal hyperalgesia. Dextrorphan (an N-methyl-d-aspartate receptor antagonist), L-703,606 (an NK-1 receptor antagonist) and an antisense oligodeoxynucleotide for a selective knockdown of protein kinase Cβ, reduced complete Freund’s adjuvant-induced hyperalgesia, and reversed significant changes in the membrane activity of protein kinase CβII on the spinal cord section ipsilateral to the inflamed paw. Dextrorphan and protein kinase Cβ antisense oligodeoxynucleotide were effective in reversing complete Freund’s adjuvant-induced increase in the activity of protein kinase CβII ipsilateral to the inflammation at all the doses tested, but L-703,606 was effective only at the highest dose. Furthermore, in the presence of inflammatory stimulus, dextrorphan and L-703,606 did not alter the activities of membrane-associated protein kinase Cα, protein kinase CβI, and protein kinase Cγ in the section of the spinal cord ipsi- and contra-lateral to the inflammation. Protein kinase Cβ antisense oligodeoxynucleotide had no significant effect on the membrane-associated activities of protein kinase Cα and protein kinase Cγ, but decreased the activities of both protein kinase CβI and protein kinase CβII and the expression of protein kinase Cβ isozyme in the spinal cord. The data provide evidence that a common molecular event that converges to initiate and maintain hyperalgesia may include the translocation and activation of protein kinase CβII in the spinal dorsal horn.
High Pressure Effects in Molecular Bioscience
2005, Chemistry at Extreme ConditionsThe limits of existence of life have been pushed to unexpected extremes of pressure, temperature, pH, salinity, etc., for the last several decades. Hydrostatic pressure significantly influences the structural properties and thus the functional characteristics of cells. However, these have not prevented the invasion of cold and high pressure habitats of deep-sea organisms. The effect of high pressure on bimolecular systems can yield a wealth of enlightening new information on their structure, energetic phase behavior and on their transition kinetics, and might promise fulfillment of the challenge set forth by W. Kauzmann when discussing the thermodynamics of unfolding of proteins. Further, pressure may also serve as valuable thermodynamic tweezers to study protein–protein interaction and aggregation. Ambitious goals that are based on the rational modification of molecular structures functions with relationships by pressure. The chapter demonstrates that pressure dependant studies can help delineate the free energy landscape of proteins and hence help elucidate which features are essential in determining the uniqueness and stability of the native conformational style. References are also given in the chapter.
Hyperalgesia induced by peripheral inflammation is mediated by protein kinase C βII isozyme in the rat spinal cord
2001, NeuroscienceWe have addressed the molecular mechanism(s) of hyperalgesia, which depends on increased excitability of dorsal horn neurons and on sensitization of primary afferent nociceptors, during peripheral inflammation. Following unilateral adjuvant-induced inflammation in the rat hind paw, time-course changes in behavioral hyperalgesia and functional activities of Ca2+/phospholipid-dependent protein kinase C isozymes were examined. Inflammation was characterized by increase in paw diameter, and behavioral hyperalgesia was quantified as paw withdrawal latency from a radiant heat source. Behavioral hyperalgesia on the injected paw was significantly increased. This was accompanied by a significant increase in total functional membrane-associated protein kinase C activity, whereas total cytosolic protein kinase C activity was unchanged on the sides of the lumbar spinal cord both contralateral and ipsilateral to the inflammation. Importantly, on the side of lumbar cord ipsilateral to the inflamed paw, the activity of membrane-associated protein kinase CβII was increased following the same time-course as the paw withdrawal latency decrease, suggesting an increased translocation of protein kinase Cβll to the membrane related to behavioral hyperalgesia. A defined mixture of purified gangliosides, which inhibits intracellular protein kinase C translocation and activation, decreased inflammation-induced paw withdrawal latency, and specifically decreased the activity of membrane-associated protein kinase Cβll on the side of the spinal cord ipsilateral to the inflammation. Quantitative immunohistochemical analyses demonstrated intensified protein kinase CβII-like immunoreactivity on the side of the spinal cord ipsilateral to the inflammation. Time-course for increases in the activity of membrane-associated protein kinase CβII, and in intensity of protein kinase CβII-immunoreactivity, paralleled inflammation-mediated changes in paw withdrawal latency and paw diameter.
Our findings indicate an apparent involvement of protein kinase CβII isozyme specifically in the molecular mechanism(s) of thermal hyperalgesia.
Regulation of phosphatidylinositide transduction system in the rat spinal cord during aging
1995, NeuroscienceAge-related functional alterations in a variety of neurotransmitter systems result in modulation of interneuronal communications which has some relevance in neurological deficits observed in the aging process. The synergistic interactions between protein kinase and inositol 1,4,5-trisphosphate (insP3)/Ca2+ pathways underlie a variety of cellular responses to external stimuli. To determine whether age-dependent changes occur in the regulation of protein kinase C and inositol 1,4,5-trisphosphate/Ca2+ pathways, insP3 contents as a marker for the release of intracellular calcium, saturation binding analysis of Ins P3 receptor using [3H]inositol 1,4,5-trisphosphate, slot/northern blot analysis of Ins P3 receptor-encoding mRNA transcripts, and the activities of Ca2+/phospholipid-dependent protein kinase C isozymes were investigated in the rat spinal cord. Inositol 1,4,5-trisphosphate content and [3H]inositol 1,4,5-trisphosphate binding site density (Bmax) were quantified in the spinal cords of young (three months old), adult (12 months old) and senescent (25 months old) male Fischer 344 rats. Spinal cord content of inositol 1,4,5-trisphosphate was increased (P 0.01) in the 25-month old compared to the three- and 12-month old animals. The density of Ins P3 receptor in particulate membranes derived from the 25-month old rats was reduced (P < 0.01), but the binding affinity (Kd) was increased (P < 0.04) by a factor of 2.2 and 3.2 at 25 months of age when compared with three- and 12-month old animals, respectively. Young and middle-aged animals showed no differences in both inositol 1,4,5-trisphosphate contents and [3H]inositol 1,4,5-trisphosphate binding site density. The quantity of Ins P3 receptor mRNA was significantly increased with age in the order 25 ≫ 12 > 3 months of age. Total functional cytosolic and membrane-associated PKC activities were decreased (P S 0.05) in the 25-month compared to the three- and 12-month old rats in which activity remained unchanged. Total membrane/cytosolic activity ratios were unchanged by the aging process. In all cases, the activities of membrane-associated conventional protein kinase C isozymes (a, β and γ), determined by immunoprecipitation followed by in situ quantification of protein kinase C activities in the immunoprecipitates, showed age-dependent decline. The activities of protein kinase C-a and β were significantly decreased in age-related manner. However, the activity of the γ-isozyme was not significantly changed at 12- and 25-months of age, although it was higher (P ≤ 0.03) in young rats. Western blot analyses using affinity purified polyclonal antibodies specific for each isozyme indicated a single protein with an apparent molecular mass of ∼ 80 × 103 molec. weight for all isozymes except for the β isozyme that also had an appreciable immunoreactive band at ∼ 36 × 103 molec. weight. Overall, the aging process did not affect the electropheretic mobility of each isozyme. With decreased protein kinase C activity, the present data suggest that the aging process would decrease protein kinase C-induced phosphorylation of membrane proteins including Ins P3 receptor. A significant change in Ins P3 receptor affinity combined with increased levels of Ins P3 receptor mRNA-encoding transcripts in senescent rats suggests not only a modifiction (possibly by phosphorylation) of Ins P3 receptor protein but also the existence of multiple (spliced) variants of Ins P3 receptor in spinal neurons with increasing age. The present data indicate that the spinal contents of inositol 1,4,5-trisphosphate increased with age, but with decreased efficacy and number of inositol 1,4,5-trisphosphate-activatable Ca2+ channels in the spinal cord of senescent rats. These age-related changes may contribute to the attenuated responsiveness of spinal cord neurons by phosphoinositide-coupled receptors during the aging process.
Long-lasting increase in protein kinase C activity in the hippocampus of amygdala-kindled rat
1995, Brain ResearchPrevious studies have demonstrated that membrane-associated protein kinase C (PKC) activities in the right and left hippocampus of rats kindled from the left hippocampus increased significantly at 4 weeks [9] and 4 months [22] after the last seizure compared with those in matched control rats. In this study, we investigated the effect of kindling from the left amygdala on PKC activities in the amygdala/pyriform cortex and hippocampus at long seizure-free intervals (4 and 16 weeks) from the last amygdala-kindled seizure. Membrane-associated PKC activity of the kindled group increased significantly only in the left hippocampus compared with the left side control (the left hippocampus of rats subjected to a sham operation) at 4 weeks (by 34%, P < 0.03) and 16 weeks (by 24%, P < 0.05) after the last seizure. There was no significant alteration in the membrane-associated PKC activity of the kindled group in the right hippocampus or amygdala/pyriform cortex in any seizure-free interval after the last amygdala seizure. Cytosolic PKC activity did not differ between the kindled and control groups in any brain region examined in any seizure-free interval. At 16 weeks after the last seizure, the PKC activity in the P1 fraction of the kindled group increased significantly only in the left hippocampus (by 49%, P < 0.005), but not in the right hippocampus. Neither PKC activity in the P2 fraction nor that in the cytosolic fraction was altered in the kindled group after this seizure-free interval. The prolonged increase in activity of the membrane-associated PKC and that in the P1 fraction in the hippocampus induced by amygdala-kindling may contribute to long-lasting seizure susceptibility induced by kindling.