Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes.

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms alpha and beta were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC alpha and PKC beta were present. In the particulate fraction two peaks of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC alpha immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC alpha whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC alpha in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC alpha and PKC beta, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG.

Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.

To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors affecting the stability and conformation of Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from the migratory locust, Locusta migratoria, represents the only full-length apolipoprotein whose three-dimensional structure has been solved. In the present study, spectroscopic methods have been employed to investigate the effects of deglycosylation (via endoglycosidase F treatment) and complexation with lipid on the stability and conformation of this protein. Addition of isolated lipid-free apoLp-III to sonicated vesicles of dimyristoylphosphatidylcholine (DMPC) resulted in the formation of relatively uniform disklike complexes with an average Strokes diameter of 13.5 nm. Flotation equilibrium experiments conducted in the analytical ultracentrifuge revealed a particle molecular mass of 588 500 Da. Chemical cross-linking and compositional analysis of apoLp-III.DMPC complexes indicated five apoLp-III molecules per disk and an overall DMPC:apoLp-III molar ratio of 122:1. Circular dichroism (CD) spectra of apoLp-III samples suggested a loss of alpha-helical structure upon deglycosylation, while complexation with DMPC did not significantly alter the helix content (estimated to be > 75%). Fluorescence spectroscopy revealed that the apoLp-III tryptophan fluorescence emission maximum was blue-shifted from 347 to 332 and 321 nm upon deglycosylation and complexation with DMPC, respectively. In quenching experiments with native apoLp-III, tryptophan residues were shielded from the positively charged quencher, CsCl. Increased exposure to KI, CsCl, and acrylamide was observed upon deglycosylation, whereas complexation with DMPC yielded lower Ksv values for KI and acrylamide and an increased value for CsCl versus native lipid-free apoLp-III. In guanidine hydrochloride denaturation studies monitored by CD or fluorescence, native, lipid-free apoLp-III displayed a denaturation midpoint of 0.60 M, and delta GDH2O = 5.37 kcal/mol was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. Evidence for a conformational change.

Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III.DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLp-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of apoLp-III.DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III.DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III.DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 M guanidine HCl, respectively. The fluorescence excitation and emission spectra of apoLp-III.DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. Denaturation of apoLp-III in the complex by guanidine HCl resulted in a tyrosine fluorescence level similar to that of lipid-free apoLp-III in the presence of guanidine HCl. The tyrosine-induced fluorescence of the complex was quenched with both Cs+ (Kq = 0.573 M-1) and KI (Kq = 0.376 M-1). The results presented in this study indicate that the conformation of apoLp-III is stabilized when complexed with phospholipids and suggest that tyrosine fluorescence provides a sensitive method to detect M. sexta apoLp-III interaction with lipid surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

A turbidimetric assay of lipid transfer activity.

A novel method to assay insect plasma lipid transfer particle (LTP) activity has been developed that employs insect high density lipophorin (HDLp) and human low density lipoprotein (LDL) as donor/acceptor substrate particles. At a 3:1 or greater HDLp:LDL protein ratio, LTP-mediated net vectorial transfer of diacylglycerol from lipophorin to LDL produces destabilized LDL particles that aggregate, causing sample turbidity. Turbidity was measured spectrophotometrically as a function of absorbance at 340 nm. After an initial lag phase, lipoprotein sample turbidity increased as a function of reaction time and LTP concentration. Saturation was observed at longer times or higher LTP concentrations, indicating that a reaction end point had been reached. As the substrate HDLp concentration was increased relative to LDL, a saturable increase in LTP-induced lipoprotein sample turbidity was observed. When the LDL concentration was increased relative to HDLp, however, there was an initial production of turbidity but at higher concentrations the sample did not develop turbidity. Reaction progress was also dependent on temperature over the range 0-37 degrees C. Taken together the results are consistent with the concept that LTP-mediated diacylglycerol transfer from HDLp to LDL creates unstable product LDL particles that aggregate. The assay method is advantageous because it employs relatively abundant, natural lipoprotein substrates, does not require prelabeling of donor lipid particles with radioactive or fluorescent lipids, and does not require separation of donor and acceptor after incubation. This is the first description of a lipid transfer assay that can be measured spectrophotometrically.

Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes.

We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca(2+)-transport and Ca2+/K(+)-ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca2+/K(+)-ATPase activity of 371 +/- 55 nmol/min/mg protein (mean +/- S.E.M.; n = 5) and an oxalate-stimulated Ca(2+)-uptake activity of 103 +/- 4 nmol/min/mg protein (mean +/- S.E.M.; n = 6). Pretreatment of the SR vesicles with 5 microM ruthenium red increased the oxalate-stimulated Ca(2+)-uptake to 204 +/- 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca(2+)-release channel), 100 (Ca2+/K(+)-ATPase), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca(2+)-binding protein bands in this preparation at 53-55 kDa (calsequestrin and/or calreticulin) and 97-100 kDa (Ca2+/K(+)-ATPase). The presence of phospholamban, a regulatory protein of the Ca2+/K(+)-ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.

Choline glycerophospholipid biosynthesis in the guinea pig heart.

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.

A sensitive method for the quantitation of lysophosphatidylcholine in canine heart.

We have developed a procedure for the determination of small amounts of lysophosphatidylcholine in cardiac tissue. Lysophosphatidylcholine from canine heart was separated from the major phospholipids by column chromatography, and then acetylated with labeled acetic anhydride. The acetylated lysophosphatidylcholine was isolated by thin-layer chromatography and the lysophosphatidylcholine content was calculated from the radioactivity associated with the acetylated product. Although the sensitivity of the assay depends on the specific radioactivity of the acetic anhydride used, as low as 0.5 nmol of lysophospholipid in tissue samples can be readily quantitated. The results obtained from the control and ischemic canine cardiac tissues by this assay compares favorably with those obtained by lipid-phosphorus assay. The sensitivity and specificity of the present procedure allows us and other investigators to assay for lysophosphatidylcholine content in very small (10 mg wet weight) tissue samples.

Plasmalogenase in hamster heart.

In this study, the presence of plasmalogenase for the hydrolysis of the alk-1-enyl bond at the C-1 position of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) in the hamster heart was examined. A new spectrophotometric assay was developed for this study, in which the aldehyde released by the hydrolysis of the plamalogenase was oxidized to carboxylic acid by the action of aldehyde dehydrogenase, with the production of the molar equivalent of NADH. The results obtained from the spectrophotometric assay were comparable to those obtained by determining the rate of ethanolamine plasmalogens utilized during the reaction. However, the sensitivity of the spectrophotometric assay for plasmalogenase was shown to be 25-fold higher than with the methods described previously and enzyme activity could be detected with 1 micrograms of microsomal protein. Hamster heart plasmalogenase activity was located exclusively in the microsomal fraction, and the enzyme displayed a pH optimum at 8.5. The enzyme showed no absolute requirement for divalent metallic cations.