Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins.

Phosphatidylinositol (PtdIns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 micrograms/ml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycan-specific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.     

Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane

We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5- to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTPgammaS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDPbetaS or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated alpha-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.     

A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors

Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca2+ approximately 5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate- and cAMP-induced Ca2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2(+)-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenyl-hydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca2+ uptake, suggest that different chemoreceptors activate a single Ca2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca2+ entry probably does not involve a Na+/Ca2+ exchanger or voltage-activated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2(+)-uptake response. Analysis of putative signaling mutants suggests that Ca2+ entry is not regulated by the guanine nucleotide-binding (G) protein subunits G alpha 1 or G alpha 2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).     

Inhibition of the plasma glycosylphosphatidylinositol-specific phospholipase D by synthetic analogs of lipid A and phosphatidic acid.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a plasma enzyme with extensive sequence similarity to integrin alpha subunits, is inhibited by micromolar concentrations of lipid A, phosphatidic acid (PA) and lysophosphatidic acid (M. G. Low and K.-S. Huang, J. Biol. Chem. 268, 8480-8490, 1993). In this study we have explored the mechanism of inhibition using synthetic analogs of lipid A, and PA. Monosaccharide analogs of lipid A, which varied in the number and position of the phosphate groups, the type of acyl group, and its linkage to the glucosamine ring, were tested for their ability to inhibit GPI-PLD. A compound (SDZ 880.431) containing 3-aza-glucosamine 1,4-diphosphate as the polar headgroup was identified which had a potency (IC(50) approximately 1 microM) similar to natural lipid A preparations. Removal of either phosphate residue increased the IC(50) markedly. Analogs of PA such as (7-nitro-2-1,3-benzoxadiazo-4-yl)amino-PA, ceramide 1-phosphate, and hexadecyl phosphate had approximately IC(50) values ranging from 1 to 5 microM, indicating that considerable variation in the structure of the hydrophobic groups was permissible. Inhibition of GPI-PLD by long-chain PA could not be blocked by high concentrations of glycerol 1-phosphate or dibutyryl PA. These results indicate that the hydrophobic groups do not have a passive role in inhibition but are directly involved in the binding interaction with GPI-PLD. We propose that this diverse group of inhibitors all bind to a common site on GPI-PLD, the central hydrophobic cavity predicted by the beta-propeller model for integrin alpha subunits and GPI-PLD.     

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.     

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host–pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.     

Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.     

A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A(1) (PLA(1)) that produces 2-acyl-LPA. The PLA(1) was identified in the GenBank(TM) data base as a close homologue of phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA(3), a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA(1) and suggest that it has a role in LPA production.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

Interaction of phospholipase A with an axon plasma membrane preparation.

The rates at which the phosphalipase A catalyzed inactivation of the axonal cholinergic binding macromolecule (ACBM), activation of acetylcholinesterase, and hydrolysis of fatty acid acyl esters were measured in an axon plasma membrane preparation from lobster nerves. The inactivation of ACBM was shown not to be caused by products of the reaction. The solubilized ACBM was also sensitive to phospholipase A inactivation. These results indicated a direct role for the phospholipid in the binding of cholinergic ligands to ACBM. It is suggested that ACBM may be a postsynaptic cholinergic receptor that is in a different lipid environment in the axon plasma membrane. A comparison with the effect of phospholipase A on axonal conduction in intact nerves suggests that the ACBM is directly involved in this process.     

Evidence that v-Src-induced phospholipase D activity is mediated by a G protein.

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca(2+)-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.     

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.     

Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane

Activities of phospholipase D (PLD) in diverse subcellular organelles have been identified but the details of regulatory mechanisms in such locations are unknown. Protein kinase C (PKC) is a major regulator of PLD. Serine 2, threonine 147, and serine 561 residues of phospholipase D1 (PLD1) were determined as sites of phosphorylation by PKC (Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., Ryu, S. H. (1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly phorbol 12-myristate 13-acetate (PMA)-induced PLD1 activity in COS-7 cells. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane (CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm, but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation, and thereby activation, of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low density domain of the plasma membrane in COS-7 cells.     

Turnover of plasma oleic acid measured by radio-gas chromatography.

Gas-liquid chromatography with radioactivity detection (Radio-GLC) was investigated as an analytical means of determining the fractional turnover rates of plasma free fatty acids. For this purpose normal dogs were infused with 1.838 muCi/min of [1-14C]oleic acid complexed with albumin and plasma samples were taken at 0 to 110 minutes. The plasma free fatty acids were isolated by a modified Dole extraction and the methyl esters, prepared by diazomethylation, were identified and quantitated by GLC and radio-GLC using radioactive methyl heptadecanoate as internal standard. The study demonstrates that physiologically feasible infusion rates and loads of radioactive acids can be found which permit accurate analyses of plasma free fatty acids by radio-GLC. During a 2-hour infusion no labeled acid other than oleic appeared in plasma indicating that the method could be used to study the turnover of a mixture of fatty acids simultaneously. These results also indicate that conventional methods of determination of radioactivity in purified extracts can be employed without concern for recycling of label among the fatty acids, at least over short periods of time. The radio-GLC technique described yields approximately 20% higher fractional turnover times for oleic acid than do standard methods.     

Association with actin mediates the EGTA-resistant binding of cytosolic phospholipase A2-alpha to the plasma membrane of activated platelets

The association of cytosolic phospholipase A₂-α (cPLA₂α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A₂ in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA₂α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA₂α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA₂α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA₂α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA₂α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA₂α with its membrane substrate via interaction with actin.     

Increase of oleic acid in erythrocytes associated with malignancies.

Total lipid extracts of erythrocyte cell membranes from 60 patients with documented malignancies, 41 patients with various acute and chronic diseases, and 40 healthy subjects were analysed. The results were expressed as ratios of stearic to oleic acid, reflecting the degree of desaturation of stearic acid. The mean ratios for the healthy subjects and controls without cancer were 1.5 (SD 0.27) and 1.45 (0.28), respectively, whereas the ratios for patients with malignancies were consistently lower than the cut off point of 1.0, with a mean of 0.69 (0.15) (p less than 0.001). The desaturation ratio was also significantly lower (p less than 0.001) in the group with recurrent tumours (mean 0.75 (0.04)) compared with those with no evidence of recurrent tumours (mean 1.55 (0.27)). It is suggested that the increased unsaturation (oleic acid) in the circulating erythrocytes may be useful in the diagnosis and postoperative monitoring of patients with cancer.     

Procalpain is activated on the plasma membrane and the calpain acts on the membrane

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.