Biomedical subjects

S Fleischer

Publications and source records attributed to S Fleischer.

At least 145 records · Page 8Linked to original sources

PubMed1987

Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes.

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.

Animals↗

PubMed1987

Noncooperative vs. cooperative reactivation of D-beta-hydroxybutyrate dehydrogenase: multiple equilibria for lecithin binding are determined by the physical state (soluble vs. bilayer) and composition of the phospholipids.

D-beta-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme that catalyzes the interconversion of beta-hydroxybutyrate and acetoacetate. The purified soluble enzyme devoid of lipid (i.e., the apodehydrogenase) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Lipid activation curves have a sigmoidal shape, and two models have been proposed to explain them. We have previously reported that the kinetics of reactivation with short-chain lecithins in the soluble state is consistent with a model in which the enzyme enzyme contains two identical, noninteracting lecithin binding sites, both of which must be occupied to activate the enzyme [noncooperative mechanism; Cortese, J.D., Vidal, J.C., Churchill, P., McIntyre, J.O., & Fleischer, S. (1982) Biochemistry 21, 3899-3908]. More recently a kinetic model involving cooperative interactions between lecithin binding sites was proposed for the reactivation of the membrane-bound enzyme [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208]. This study reinvestigates the basis for the different conclusions in these two studies. The previous study with soluble lecithins was limited to about 34% of maximal activation compared with mitochondrial phospholipid, due to inactivation of the enzyme at the critical micellar concentration. We could now extend this study to 91% activation by increasing the ethanol concentration. This experimental evidence confirms that the soluble system follows a noncooperative equation. We provide a new kinetic approach to test the cooperative model. A velocity equation is derived for a Hill-type cooperative ligand binding system interacting with a mixture of ligands. This equation predicts a proportionality between an overall weighted cooperative dissociation constant [Kcoop(w)] and a dissociation constant for a single lecithin (PC) species from interacting sites (KPC), regulated by the PC molar fraction (XPC): 1/Kcoop(w) = XPC/KPC. The equation was applied to the data of Sandermann et al. [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208] as well as to newly obtained data. The results obtained over a wide range of PC molar fractions and different mixtures of bilayer phospholipids fit this equation, confirming the cooperative behavior. We conclude that BDH has a different mode of reactivation depending on the nature of the lipid environment. With soluble lecithin, the activation is noncooperative, whereas in the bilayer, mixtures of phospholipids give cooperative behavior that fits a Hill equation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Target size of the ryanodine receptor from junctional terminal cisternae of sarcoplasmic reticulum.

Target inactivation analysis was carried out on the ryanodine receptor. This receptor recently has been implicated as the channel involved in the calcium release process in excitation-contraction coupling and was localized to the junctional terminal cisternae of sarcoplasmic reticulum from skeletal muscle [Fleischer, S., Ogunbunmi, E. M., Dixon, M. C., & Fleer, E.A.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7256-7259]. Irradiation of the junctional terminal cisternae resulted in an exponential decrease in ryanodine binding with radiation dose, thereby consistent with target theory. The target molecular weight was found to be 138,000 +/- 21,000, i.e., smaller than the polypeptide that binds ryanodine. The calcium pump protein in the same membrane preparation served as an internal control to validate the methodology.

Animals↗

PubMed1987

Distance estimate of the active center of D-beta-hydroxybutyrate dehydrogenase from the membrane surface.

D-beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) is a membrane-bound, lipid-requiring enzyme which has a reactive sulfhydryl in the vicinity of the active center. The spin-probe-spin-label technique has been used to estimate the distance of separation of the reactive sulfhydryl of D-beta-hydroxybutyrate dehydrogenase from the bilayer surface. The reactive sulfhydryl of the enzyme was derivatized with the maleimide spin-label reagent 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy in the presence of the cofactor NAD+. The derivatized enzyme, inserted (inlaid orientation) into phospholipid vesicles, was titrated with spin probes, either Mn2+ or Gd3+, until the spin-label EPR spectrum was reduced in amplitude to its residual (limiting) value. From this limiting amplitude, the dipolar interaction coefficient was obtained, which is related to the reciprocal of the distance to the sixth power. The radial distances of closest approach of the paramagnetic Mn2+ and Gd3+ ions to the spin-label nitroxide on the enzyme were found to be 18 and 16 A, respectively. These calculated distances were in accord with those determined by comparison with a phosphatidylcholine calibration system having 2,2-dimethyloxazolidinyl-1-oxy spin-labels located at selected positions along the sn-2 fatty acyl chain. Since the distal nitroxide moiety of the maleimide spin-label (17 A from the bilayer surface) is 8 A from the sulfhydryl addition site, the two limiting distances of immersion of the reactive sulfhydryl within the bilayer are 9 and 25 A. The shorter distance is considered more compatible with facile access of the coenzyme to the active site of the enzyme.

Binding Sites↗

PubMed1987

Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle.

The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.

Animals↗

PubMed1987

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle.

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.

Animals↗

PubMed1986

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum.

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1986

Apparent cooperativity of Ca2+ binding associated with crystallization of Ca2+-binding protein from sarcoplasmic reticulum.

Needle-shaped crystals of the Ca2+-binding protein (CBP) isolated from rabbit skeletal muscle sarcoplasmic reticulum were studied with regard to the influence of Ca2+, K+, and H+ on its solubility and cation binding. The solubility of CBP is sharply decreased with concentration of Ca2+, whereas K+ increased it. Aggregation of the CBP and crystal formation is correlated with the binding of Ca2+. The Ca2+ bound to the crystalline CBP is two to three times higher than that of the soluble form. A strong apparent positive cooperative behavior of Ca2+ binding by CBP was observed concomitant with the shift in equilibrium from the soluble to the crystalline form. From the steepest Hill slope we obtained Hill coefficients of 3.3 for soluble CBP and 14 for the transition between soluble and crystalline forms of CBP. A detailed treatment is presented to validate the applicability of Hill plots for the combined binding and crystallization process. Two-thirds of the Ca2+-binding sites were K+ sensitive and one-third were K+ insensitive. An increase in H+ concentration decreased the Ca2+ binding by crystalline CBP without affecting its solubility, with a pK value of 6.2 determined for this process. These results indicate that the equilibrium between the soluble and crystalline forms of CBP is determined by the amount and nature of the bound cations, Ca2+, K+, and H+. They suggest the possibility that a cycle of aggregation and solubilization of CBP attends the uptake and release of Ca2+ in the sarcoplasmic reticulum, respectively.

Animals↗

PubMed1986

The secondary structure of calcium pump protein in light sarcoplasmic reticulum and reconstituted in a single lipid component as determined by Raman spectroscopy.

Raman spectra have been measured of the following samples: active calcium pump protein in light sarcoplasmic reticulum (SR) membranes, lipids extracted from light SR membranes, active calcium pump protein reconstituted in dielaidoylphosphatidylcholine (DEPC), and pure DEPC. The spectra of native SR lipids and of pure DEPC are different, and yet when these spectra are subtracted from the spectra of the respective protein-lipid complexes, the resulting amide I spectra of the calcium pump protein are the same, indicating that appropriate criteria have been chosen for subtraction of the spectrum of a lipid. This spectrum has been analyzed for secondary structure with the following results. The SR calcium pump protein contains 51 +/- 5% helix, in agreement with a prediction of secondary structure obtained from an analysis of the sequence, and 21 +/- 4% beta-strand. In addition, the presence of protein broadens and lowers the main melting transition of DEPC.

Animals↗

PubMed1986

Characterization of polyisoprenyl phosphate phosphatase activity in rat liver.

The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis-polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm.

Animals↗

PubMed1986

Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin.

Binding Sites↗

PubMed1986

The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum.

Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 +/- 0.31 mumol mg-1 min-1, Ca2+-activated ATPase activity = 2.39 +/- 0.25 mumol mg-1 min-1, efficiency (Ca2+/ATP) = 0.69 +/- 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KCa) in the original vesicles from 1.35 +/- 0.08 microM to 0.75 +/- 0.12 microM by cAMP-dependent phosphorylation of phospholamban. On the other hand KCa for the reconstituted vesicles was about 0.5 microM and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.

Animals↗

PubMed1986

Detection of rearranged T cell receptor beta-chain gene and induction of cytolytic function in interleukin 2-responsive day 14-15 murine fetal thymocytes.

A subpopulation of interleukin 2 (IL 2) receptor-positive day 14-15 murine fetal thymocytes can be induced by recombinant IL 2 to proliferate over prolonged time periods in dissociated cell cultures. The proliferating day 14-15 fetal thymocytes exhibit no cytolytic effector function, nor do they rearrange T cell receptor beta chain genes. This contrasts with thymic organ cultures in which day 14-15 thymocytes do rearrange beta chain genes and give rise to immunocompetent cells. However, such events can also take place in dissociated cell cultures, provided the IL 2-responsive thymocytes are cultured on syngeneic feeder cells in the presence of IL 2 and the mitogen concanavalin A. Under such conditions rearrangement of the beta chain gene complex becomes detectable and cytolytic effector cells are generated. The frequency of inducible cytolytic precursor cells in day 14-15 thymocytes is 1/7000. These data either imply that immunocompetent cells are already present in the day 14-15 fetal thymus, or differentiation from precursors to immunocompetent cells must occur in dissociated cell cultures.

Animals↗

PubMed1986

Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum.

We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85% of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles. Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5% Triton X-100 in the presence of 1 mM CaCl2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 mumol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca2+ release process in excitation-contraction coupling, is now available in the test tube.

Animals↗

PubMed1986

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis.

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.

Animals↗

PubMed1985

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction.

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Animals↗

PubMed1985

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Purification and crystallization of the calcium binding protein of sarcoplasmic reticulum from skeletal muscle.

The calcium binding protein of skeletal muscle sarcoplasmic reticulum (also referred to as calsequestrin) was purified by release from the compartment of the vesicles with the detergent octaethyleneglycol mono-n-dodecyl ether (C12E8) and by subsequent precipitation of the calcium binding protein with specific divalent cations. The isolated protein exhibited a single band on NaDodSO4/PAGE and bound 903 nmol of Ca2+ per mg of protein. The calcium binding protein could be crystallized in the presence of Ca2+, Mg2+, Sr2+, or combinations of these three cations used in a narrow concentration range. Needle-shaped crystals of up to 500 X 50 micron were obtained. The removal of the divalent cations resulted in solubilization of the crystals. The spacings and angles of the crystals were obtained by electron microscopy using three different methods of sample preparation. By freeze-drying and negative staining electron microscopy, the spacings along axes a and b were determined to be 10-11 nm each, and the angle between the two axes was 90 degrees. By thin section electron microscopy, the spacing along axis a was 11 nm, along axis c was 15-16 nm, and the angle between the two axes was 75 degrees. This study reports (i) a simple and rapid method for purification of the calcium binding protein; (ii) conditions to crystallize the protein using Ca2+, Mg2+, Sr2+, or combinations of the three; and (iii) some preliminary characteristics of the crystals. The crystalline nature was characterized by electron microscopy and x-ray diffraction. The larger crystals diffracted beyond 3-A Bragg spacing.

Animals↗