Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37°C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.
In this work we compared the effect of five heavy metals: Zn, Pb, Cd, Ni and Cu on phospholipid composition of the ubiquitous soil fungus Paecilomyces marquandii, originating from a strongly metal polluted area and characterized by high tolerance to these elements. Cd, Ni and Cu caused an increase in phosphatidylcholine (PC). Only Pb decreased PC content, which was accompanied by a significant rise in the phosphatidic acids (PA) level, probably due to activation of phospholipase D which hydrolyzes PC to PA. This could result in membrane fluidity disturbance, and thus affect its integrity. The assessment of propidium iodide influx showed strong disturbance of membrane integrity for Cu and Pb stressed mycelia, whereas mycelia treated with Ni were impermeable to this dye. The results obtained revealed a strong Cu and Pb toxicity involving disruption of membrane integrity. Pb action was reflected by lipid composition, whereas changes in Cu treated mycelia did not completely elucidate its harmful effect on the membrane, which was most probably caused by Cu induced lipid peroxidation. Zn did not induce quantitative changes in PC and phosphatidylethanolamine (PE) but caused changes in phospholipid lipid saturation, which appears to be important for fungus adaptation to the presence of metals. The enhanced PC content balanced by higher PC saturation can help in the maintenance of proper membrane fluidity and result in alleviating the Cd and Ni induced stress. These results will allow to clarify the mechanism of Pb toxicity and help to elucidate the cellular basis of fungal membrane adaptation to heavy metals.
In plant cells, phospholipids are not only membrane components but also act as second messengers interacting with various proteins and regulating diverse cellular processes, including stress signal transduction. Here, we report studies on the effects of various phospholipids on the activity and expression of maize wound-responsive calcium-dependent protein kinase (ZmCPK11). Our results revealed that in leaves treated with n-butanol, a potent inhibitor of phosphatidic acid (PA) synthesis catalyzed by phospholipase D, a significant decrease of ZmCPK11 activity was observed, indicating contribution of PA in the kinase activation. Using lipid binding assays, we demonstrate that among various phospholipids only saturated acyl species (16 : 0 and 18 : 0) of phosphatidic acid are able to bind to ZmCPK11. Saturated acyl species of PA are also able to stimulate phosphorylation of exogenous substrates by ZmCPK11 and autophosphorylation of the kinase. The level of ZmCPK11 autophosphorylation is correlated with its enzymatic activity. RT-PCR analysis showed that transcript level of ZmCPK11 in maize leaves increased in response to PA treatment. The influence of PA on the activity and transcript level of ZmCPK11 suggests an involvement of this kinase in a PA-mediated wound signal transduction pathway.
The etiology of altered blood fatty acid (FA) profile in phenylketonuria (PKU) is understood only partially. We aimed to determine whether FAs deficiency is dependent on the diet or metabolic disturbances. The study comprised 40 PKU patients (20 female, 20 male; aged 11 to 35 years; 12 children and 28 adults) and 40 healthy subjects (HS; 20 female, 20 male, aged 18 to 33 years). We assessed the profile of FAs (gas chromatography/mass spectrometry) and analyzed the 72-hour dietary recalls. The amount of C14:0, C16:0 and C16:1n-7, C18:1n-9 did not differ between the analyzed groups. The percentage of C18:0 was higher, while C20:3n-9, C18:2n-6, C20:2n-6, C20:4n-6, C22:4n-6, C22:5n-6 and C22:6n-3 was lower in PKU than in HS. However, C18:3n-6, C18:3n-3 and n-6/n-3 ratio were higher in PKU patients. The C20:4n-6/C20:3n-6 ratio (reaction catalyzed by Δ5-desaturase), the C22:5n-6/C22:4n-6 and the C22:6n-3/C22:5n-3 ratio (both reactions catalyzed by Δ6 desaturase) were significantly lower in PKU patients. Therefore, the deficiency of long-chain polyunsaturated fatty acids in PKU patients may result not only from inadequate supply but also from metabolic disturbances.
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