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Biomedical subjects

S Fleischer

Publications and source records attributed to S Fleischer.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of an exchange reaction between soluble FKBP-12 and the FKBP.ryanodine receptor complex. Modulation by FKBP mutants deficient in peptidyl-prolyl isomerase activity.

FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK-590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S. (1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S. (1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP.RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 microM for wild type FKBP versus 0.6 to 2.4 microM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP.RyR complex is in equilibrium with the cytosolic pool of FKBP (approximately 3 microM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity.

Amino Acid Isomerases↗
PubMed1995

Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle.

The cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with highly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channel block by Mg2+ (-mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg2+ concentrations (-mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca2+ contraction cycle of the heart. For example, Ca2+ release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg2+. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.

Animals↗
PubMed1995

Phosphorylation with protein kinases modulates calcium loading of terminal cisternae of sarcoplasmic reticulum from skeletal muscle.

We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg2+ when terminal cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca2+/calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823-1833]. In this study, modulation by protein kinases and phosphatases on net Ca2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 mM MgCl2. There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incorporation of [gamma-32P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 +/- 0.1 (32P/RyR) for PKA, 0.89 +/- 0.08 for CamPK II and 0.95 +/- 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg2+] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Imidodiphosphate↗
PubMed1995

IP3 receptor purified from liver plasma membrane is an (1,4,5)IP3 activated and (1,3,4,5)IP4 inhibited calcium permeable ion channel.

The IP3 receptor is involved in Ca2+ mobilization from intracellular stores. Recently, we purified an inositol (1,4,5)-trisphosphate receptor from rat liver plasma membrane (LPM-IP3R) [Schäfer R. Hell K. Fleischer S. (1993) Purification of an IP3 receptor from liver plasma membrane. Biophys. J. 66, A146]. The purified LPM-IP3 receptor was incorporated into vesicle derived planar bilayers and its channel properties characterized. The receptor displayed ion channel activity that was activated by inositol (1,4,5)-trisphosphate [(1,4,5)IP3] (1 microM) and inhibited by inositol (1,3,4,5)-tetrakisphosphate (IC50 approximately 1 microM) and by heparin (IC50 approximately 20 micrograms/ml). The channel displays a unitary conductance of 9 pS, and 13 pS in symmetrical 100 mM and 500 mM KCl, respectively, and in symmetrical 250 mM cesium methanesulfonate the slope conductance is 11 pS. Activation by (1,4,5)IP3 is specific to the cis-side of the chamber, equivalent to the cytoplasmic face. The receptor is a Ca2+ permeable ion channel based on ion selectivity (Ca2+ > K+ > Na+ >> Cl). The LPM-IP3 receptor was also permeable to Cs (Cs+ > or = K+), similar to other intracellular Ca2+ release channels, i.e. the IP3 receptor from brain and smooth muscle (IP3R-1) and the ryanodine receptor from skeletal muscle (RyR-1) and heart (RyR-2). Channel activity is not voltage dependent (+/- 100 mV applied voltage). The channel is activated by ATP and Ca2+. The open probability of the (1,4,5)IP3 activated channel activity displays a bell shaped response to cis Ca2+ ion concentration of our system. The LPM-IP3 receptor differs from intracellular IP3R-1 in that the Ca2+ and ATP concentration required for maximum activation is about 10 times higher as compared with IP3R-1 from brain cerebellum and smooth muscle. We conclude that the LPM-IP3 receptor is an (1,4,5)IP3 activated Ca2+ permeable ion channel. The implication of our studies is that in liver, (1,4,5)IP3 regulates Ca2+ influx via the plasma membrane.

Adenosine Triphosphate↗
PubMed1995

Site-specific integrated pest management for high value crops: sample units for map generation using the Colorado potato beetle (Coleoptera: Chrysomelidae) as a model system.

Site-specific agriculture uses maps to optimize within-field placement of agricultural practices. This technology introduces the potential to optimize pest management by varying pesticide or other inputs to better match within-field variation in pest density. Current sampling plans are designed to estimate mean density and may not be suitable for mapping, although useful sampling plans could be developed for map generation for integrated pest management. Using Colorado potato beetle, Leptinotarsa decemlineata (Say), adults, larvae, and egg masses as model systems, the influence of the sample unit on map validity was explored. Adapting currently used sampling plans for potato integrated pest management by spatially referencing each sampled stem failed to resolve spatial dependence and resulted in maps with poor reliability. Increasing the sample unit improved resolution of spatial dependence and map reliability for each life stage. A distance-walk sample unit for adult and late instar Colorado potato beetles which has high potential for map generation is introduced. Using this sample unit, generalizations about Colorado potato beetle spatial dependence are made to discuss issues of developing sampling programs for map generation. An iterative process of sampling, spatial analysis, and error analysis is suggested for evaluating sample units for mapping pest density in high value crops.

Animals↗
PubMed1994

Golgi coatomer binds, and forms K(+)-selective channels gated by, inositol polyphosphates.

Coatomer is a distinct type of coat protein complex involved in the formation of specific Golgi intercisternal transport vesicles. Direct binding studies using purified coatomer isolated from bovine liver cytosol show that coatomer specifically binds both inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4) and inositol hexakisphosphate (IP6) with subnanomolar affinities (0.1 and 0.2 nM, respectively). Diphosphoinositol pentakisphosphate (PP-IP5) is an efficient competitor for both (1,3,4,5)IP4 and IP6 binding to coatomer. Inositol 1,3,4,5,6-pentakisphosphate ((1,3,4,5,6)IP5) is a poor inhibitor of IP6 binding, whereas little or no competition is detected with inositol 1,4,5-trisphosphate ((1,4,5)I-P3). Coatomer displays ion channel activity when reconstituted into planar bilayers which is preferentially permeable to K+. Permeability ratios of the channel are PK+/PCl- approximately 8.0 and PK+/PNa+ approximately 7.1, indicating a cation-selective channel with selectivity of K+ over Na+. In symmetrical 500 mM KCl, the smallest observable unitary channel conductance is 8.3 picosiemens. The coatomer channel activity is normally active with long open times (0.1 to several seconds) and is selectively blocked by 10 microM (1,3,4,5)IP4, 1 microM IP6, and 0.27 microM PP-IP5; even lower concentrations are sufficient to induce channel flicker. The channel activity is not affected by (1,4,5)IP3, or (1,3,4,5,6)IP5. Thus, the channel activity of coatomer is modulated by the inositol polyphosphates which exhibit tight binding to the complex.

Animals↗
PubMed1994

The ryanodine receptor from canine heart sarcoplasmic reticulum is associated with a novel FK-506 binding protein.

We find that the purified ryanodine receptor/calcium release channel (CRC) isolated from canine heart sarcoplasmic reticulum (SR) is tightly associated with an FK-506 binding protein which is distinct from human recombinant FKBP-12. Fractions from both skeletal and heart muscle were probed by Western Blot analysis using sequence specific antibody raised against the N-terminus of human recombinant FKBP-12. Only FKBP-12 was detected in rabbit and dog skeletal muscle SR as well as in dog heart cytosol. Canine heart SR and the purified canine heart ryanodine receptor contained an immunoreactive band with a somewhat slower relative mobility than that of human recombinant FKBP-12. These observations indicate that the association between the cardiac CRC and this novel FK-506 binding protein is specific.

Animals↗
PubMed1994

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor.

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

Animals↗
PubMed1994

Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.

Adenosine Triphosphate↗
PubMed1994

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.

Animals↗
PubMed1994

Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle.

The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 degrees rotation about an axis in the plane of the specimen grid. The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca(2+)-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.

Animals↗
PubMed1993

Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.

Animals↗
PubMed1993

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein. Dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum.

The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.

Animals↗
PubMed1993

A cardiac clathrin assembly protein forms a potassium channel in planar lipid bilayers.

A novel clathrin assembly protein (designated cardiac AP-3) has been isolated from dog heart which forms a K+ channel in planar lipid bilayers. AP-3 facilitated the in vitro formation of clathrin cages, which is diagnostic for clathrin assembly proteins. AP-3 consists mainly of 100-, 97-, and 55-kDa bands. A GTP-binding protein of approximately 25 kDa also co-purifies. The 100-kDa band was recognized by a monoclonal antibody to the gamma-adaptin of bovine brain clathrin assembly protein AP-1. A polyclonal antibody to the approximately 100-kDa doublet (alpha- and beta-adaptins) of bovine brain AP-2 did not cross-react with the purified protein. Western blot analysis of cardiac subcellular fractions showed that anti-AP-1 immunoreactivity was strongest in a sarcolemma-enriched fraction. Little immunoreactivity was detected in other cardiac subfractions, including sarcoplasmic reticulum, intercalated discs, and mitochondria. When reconstituted into planar lipid bilayers, AP-3 displays ion channel activity. Permeability ratios were PK/PCl approximately 16 and PK/PNa approximately 3, indicating a cation-selective channel somewhat selective for K+ versus Na+. The K+ channel displays several subconductance states (9 and 12 picosiemens in the main) and was blocked by CaCl2 (mM), inositol 1,3,4,5-tetrakisphosphate (20 microM), inositol 1,4,5-trisphosphate) (40 microM), and guanosine 5'-O-(3-thiotrisphosphate) (mM). Thus, the cardiac AP-3 appears to act as a K+ channel modulated by inositol polyphosphates and a small GTP-binding protein.

Adaptor Proteins, Vesicular Transport↗
PubMed1993

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme.

Monoclonal antibodies (mAbs) have been used to study structure-function relationships of (R)-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mAbs to conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleavage, indicative of a PC-induced conformational change in the enzyme.

Animals↗
PubMed1993

Different intracellular localization of inositol 1,4,5-trisphosphate and ryanodine receptors in cardiomyocytes.

The ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors have previously been found to be intracellular Ca2+ release channels characterized by their large size and 4-fold symmetry. In this study, cardiomyocytes are found to have a different intracellular localization for the two receptors. At the level of light microscopy, the IP3 receptor is immunolocalized in rat ventricular cardiomyocytes at the region of the intercalated discs. By contrast, immunoreactivity of the ryanodine receptor is observed as transverse bands throughout the length of the cardiomyocyte, coincident with the triad junction at the I-bands. At the level of electron microscopy, immunogold particles directed to the IP3 receptor specifically decorate the intercalated discs of rat ventricular and atrial cardiomyocytes, preferentially at the fascia adherens. Binding of [3H] IP3 and [3H]ryanodine were measured in cardiac subcellular fractions. IP3 binding is enriched in a fraction containing intercalated discs. Little or no IP3 binding was detected in longitudinal sarcoplasmic reticulum (SR), junctional SR, sarcolemma, mitochondria, and submitochondrial vesicles. Ryanodine binding is the highest in junctional SR. We conclude that the IP3 receptor is present in ventricular and atrial cardiomyocytes and localized at the region of the intercalated discs. These results suggest a possible role of the IP3 receptor in Ca2+ entry through intercalated discs and/or intercellular signaling between cardiomyocytes.

Animals↗
PubMed1993

Anthracycline-induced tension in permeabilized cardiac fibers: evidence for the activation of the calcium release channel of sarcoplasmic reticulum.

Anthracyclines, such as doxorubicin (DOX), are important cancer chemotherapeutic agents that are cardiotoxic. The mechanism for the cardiotoxicity is not well-defined. Recent studies have concluded that anthracyclines release calcium (Ca2+) from membrane fractions containing sarcoplasmic reticulum (SR). To determine whether anthracyclines release Ca2+ in situ from cardiac SR, the effects of DOX on Ca(2+)-activated contractions were analyzed in membrane-permeabilized and membrane-intact fibers from rabbit heart. DOX (10-120 microM) induced tension development in calcium-preloaded permeabilized fibers. DOX-induced tension required submicromolar Ca2+, and was blocked by ruthenium red (20 microM) and Triton X-100 treatment, characteristics shared by caffeine-induced tension referable to SR Ca(2+)-release. DOX (50 microM) did not alter the maximum Ca(2+)-activated tension or shift the Ca2+ concentration-tension relationship of permeabilized fibers, indicating no effect of DOX on the myofilaments. DOX (44-350 microM) depressed post-rest isometric contractility of membrane-intact fibers but did not inhibit steady-state contractility (at 1 Hz; 2.5 Mm Ca2+), similar to effects of caffeine and submicromolar ryanodine. The specific effects of DOX on post-rest contractility of membrane-intact fibers are consistent with DOX-induced Ca2+ release from the SR of membrane-permeabilized fibers. Thus, DOX alters SR Ca2+ release in situ which may contribute to the inotropic and lusitropic dysfunction observed with anthracyclines.

Animals↗