Biomedical subjects

J F Marecek

Publications and source records attributed to J F Marecek.

At least 19 recordsLinked to original sources

PubMed2001

The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release.

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.

Adenosine Triphosphatases↗

PubMed1998

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Biocompatible Materials↗

PubMed1996

Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor.

A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12% of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinase-catalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.

Amino Acid Sequence↗

PubMed1993

Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands.

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.

Affinity Labels↗

PubMed1993

Synthesis and use of fluorescent molecular probes for measuring cell-surface enzymatic oxidation of amino acids and amines in seawater.

A method for investigating cell-surface enzymatic oxidative deamination of amino acids and amines in seawater was developed. This technique used synthetic fluorescent Lucifer Yellow derivatives of the amino acid lysine and the amine cadaverine as molecular probes to investigate oxidation pathways and rates. The probes were chemically stable under the conditions used and did not adsorb to container surfaces. The oxidative deamination of the fluorescent probes added to phytoplankton cultures and the subsequent production of their fluorescent oxidation products could be selectively detected by HPLC at 250 pM levels. This approach allows selective investigation of cell-surface enzymatic oxidation since neither transport of the probes across the cell membrane nor chemical transformation of the probes occurs. Bacteria were also capable of oxidizing the fluorescent amino acid probe.

Adsorption↗

PubMed1992

Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.

The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.

Animals↗

PubMed1992

New tetherable derivatives of myo-inositol 2,4,5- and 1,3,4-trisphosphates.

(+/-)-myo-Inositol 1-(3-aminopropyl hydrogen phosphate) 3,4-bis(disodium phosphate) (5) and (+/-)-myo-inositol 2-(3-aminopropyl hydrogen phosphate) 4,5-bis(disodium phosphate) (11) have been synthesized by conventional procedures. Each derivative has been immobilized on a polymeric resin in order to give a bioaffinity matrix.

Inositol Phosphates↗

PubMed1992

Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins.

We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.

Affinity Labels↗

PubMed1992

Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia.

Inositol 1,4,5-trisphosphate (InsP3), a product of G-protein-mediated receptor activation of phosphoinositide turnover, plays the role of a second messenger when olfactory neurons are stimulated with certain olfactory stimuli. In this paper we examine the specific binding of [3H]InsP3 to isolated olfactory cilia, microsomes and brain membranes from the channel catfish (Ictalurus punctatus) and, by photoaffinity labelling with an InsP3 analogue (125I-labelled 1-[3-(4-azidosalicyloxy)-aminopropyl]inositol 1,4,5-trisphosphate (125I-ASA-InsP3)], we tentatively identify the major InsP3-binding protein in catfish olfactory cilia. InsP3 binding to ciliary membranes is specific and saturable, with a Kd of 1.10 +/- 0.31 microM and a maximum number of binding sites (Bmax) of 17.6 +/- 5.8 pmol/mg. The rank order for potency of inhibition of [3H]InsP3 binding is Ins(1,4)P2 less than Ins(1,3,4)P3 less than Ins(1,3,4,5)P4 = Ins(1,4,5)P3 less than Ins(2,4,5)P3. Exposure of cilia membranes to u.v. light in the presence of 125I-ASA-InsP3 results in the labelling of a protein with apparent Mr 107,000. Labelling is specifically prevented by Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(1,3,4,5)P4, but not by Ins(1,4)P2 or Ins(1,3,4)P3. Both specific [3H]InsP3 binding and photoaffinity labelling of the Mr-107,000 protein were displaced by heparin. The Kd and the inhibition of [3H]InsP3 binding and of photoaffinity labelling by inositol phosphates and heparin are consistent with the ability of micromolar concentrations of Ins(1,4,5)P3 [but not Ins(1,3,4)P3] to activate the InsP3-gated currents in patch-clamp experiments with olfactory neurons. These results suggest that InsP3 binding to a Mr-107,000 cilia membrane protein may represent binding to the olfactory InsP3-gated cation channel.

Animals↗

PubMed1992

Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia.

Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP3). We have characterized the binding of [3H]InsP3 to isolated ROC. Unlabeled InsP3 displaced [3H]InsP3 binding in a dose-dependent manner (dissociation constant = 3.9 +/- 0.65 microM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the 125I-labeled InsP3 analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phospho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [3H]InsP3 to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D-myoinositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP3-gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP3 binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP3 concentration in rat olfactory cilia is at a ciliary InsP3-gated channel.

Animals↗

PubMed1988

Two pathways for oxygen exchange by heavy meromyosin and their dependence on actin.

In the hydrolysis of MgATP by acto heavy meromyosin (HMM) there are two enzymatic pathways that differ in the properties of their intermediate oxygen exchange; one of these is designated the low exchange pathway (P1); the other is designated the high exchange pathway (P2). A plot of the P1 flux versus the actin concentration gives a sigmoid curve, whereas the corresponding curve for the P2 flux rises in an approximately hyperbolic manner. At low concentrations of actin, where the sigmoid curve of the P1 flux is in a lag phase, the major flux is along P2; but at higher concentrations of actin, as the P1 curve rises sharply, the flux along P1 comes to predominate. Even at the highest levels of actin, at saturating levels for both pathways, the kinetics of exchange along P1 and P2 are significantly different. In addition to these differences in the actin dependence, the flux of P1 relative to P2 is markedly inhibited by KCl. Therefore, which of the two pathways dominates during the hydrolysis of MgATP by HMM is strongly dependent on experimental conditions. The findings suggest that P1 involves the interaction of HMM with two actin units whereas P2 involves the interaction of HMM with one actin unit. The results are discussed in relation to a kinetic scheme based on this proposal.

Actins↗

PubMed1986

The two pathways for oxygen exchange by actomyosin and myofibrils and their dependence on temperature.

At an intermediate stage in the hydrolysis of MgATP by actomyosin there is an exchange of oxygen between water and the terminal phosphoryl group of MgATP, tightly bound to the myosin active site. This intermediate oxygen exchange results from the reversible hydrolysis of the bound MgATP. The rate of the exchange cycle (hydrolysis and the reverse) is assumed to be determined by the rate of reverse hydrolysis; and the average time available for exchange is determined by the post-exchange reaction that immediately follows the cycle. Past analytical studies of the exchange, using actomyosin mixtures and myofibrils at room temperature, have revealed two pathways for hydrolysis, operating at a comparable flux but differing greatly in the extent of exchange they support. It is shown here that these pathways also appear over a range of temperatures from 5 to 30 degrees C and that temperature had little effect on their relative fluxes. At each temperature, the flux ratio (%) for the low exchange pathway: high exchange pathway was near 50:50 for actomyosin mixtures and 60:40 for myofibrils. Apparently, the rate-limiting steps that determine the fluxes of the two pathways have a similar temperature dependence. However, the analysis indicates that one or both of the steps that determine the extent of exchange (reverse-hydrolysis and/or the post-exchange reaction) shows a different temperature dependence for the two pathways. We interpret this to reflect a difference in the temperature dependence of the post-exchange reaction, which we propose is exceedingly fast and independent of actin concentration along the low exchange route, but slow and dependent on the actin concentration along the high exchange route. Thus at all temperatures over a broad range of actin concentration there are two pathways of comparable flux that differ primarily in the time available for exchange.

Actins↗

PubMed1986

Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.

In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A2 on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A2 on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30 degrees C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.

Calcium↗

PubMed1986

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2.

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.

Anions↗

PubMed1986

The affinity of phospholipase A2 for the interface of the substrate and analogs.

In the intravesicle scooting mode of interfacial catalysis, the interfacial complex E*S is formed by the interaction of the membrane bound phospholipase A2 (E*) with the substrate monomer (S) in the interface. In the presence of nonhydrolyzable substrate analogs (I) the kinetics of interfacial catalysis is modified. If phospholipase A2 is added to a mixture of the vesicles of L-DMPMe ester and of DTPMe ether or D-DMPMe ester, the extent of hydrolysis, A, decreases and the interfacial scooting rate constant, ki, remains unchanged. On the other hand, when the enzyme is added to the vesicles prepared from premixed L-DMPMe ester with D-DMPMe ester or L-DTPMe ether, ki decreases but A remains constant. Qualitatively, these results are in excellent accord with the Scheme I for interfacial catalysis. However, a quantitative departure has been noted, which suggests that the interfacial dissociation constant for E*S is larger than that for E*I. These results are interpreted to suggest that the catalytic rate constant for decomposition of E*S to E* + P is larger than the rate constant for decomposition of E*S to E* + S. Broader implications of the scooting mode of interfacial catalysis are discussed.

Catalysis↗

PubMed1984

Lateral interaction of cholesterol in diacylphosphatidylcholesterol bilayers.

Thermotropic phase-transition properties of the aqueous dispersions of several diacylphosphatidylcholesterol (DRCh) analogs are examined. The aqueous dispersions of their calcium salts exhibit characteristic endothermic thermotropic transitions due to a change in the conformation of acyl chains. These dispersions consist of osmotically intact liposomes that trap ions, and at the transition temperature there is anomalous increase in the ion leakage. Wide-angle electron diffraction studies of DPCh . Ca monolayers also exhibit a transition from a sharp 4.25 A band to a broad one centering at 4.7 A, reflecting an order-disorder transition in the acyl chains. The long-range order in the organization of acyl chains of DRCh molecules could arise from intermolecular interactions between the cholesterol moieties to form a functional dimer, and such dimers are apparently cross-linked by Ca2+ to form a long-range interacting lattice of acyl chains. Evidence for this model is adduced from the fluorescence properties of the dispersions of dimyristoylphosphatidylcholesta-5,7,9-trienol. The phase-transition properties of DRCh are an ideal illustration of calcium-induced isothermal phase transition.

Cholestanes↗

PubMed1984

Evidence from oxygen exchange studies that the two heads of myosin are functionally different.

Recent studies of oxygen exchange have shown that there are two normal pathways for the hydrolysis of MgATP by myosin in the presence of actin, each producing Pi at the same rate. These two apparent pathways for actin-activated hydrolysis differ greatly in the extent of oxygen exchange they support. This is revealed by an analysis of the distribution of [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. We have extended these studies to certain abnormal substrates, using Mn2+ in place of Mg2+, and dATP or ITP in place of ATP. The results, together with past findings, lead to the proposal that the two heads of myosin are functionally different. One of these (Head 1) is able to reversibly cleave bound MgATP and thereby support oxygen exchange while it is free of actin; the other (Head 2) cannot cleave bound MgATP at its active site while free of actin. However, in the presence of actin, both Head 1 and Head 2 cleave bound MgATP, support some oxygen exchange, and produce Pi at the same rapid actin-activated rate. Apparently, MgATP is positioned differently on the two heads when they are free of actin, with Mg2+ and the 6-amino group of ATP playing an important role in the orientation. This proposed difference between the heads could serve to make Head 1 react first with the actin filament, followed by Head 2. Thus, in muscle, the two heads on a myosin cross-bridge would interact with an actin filament and exert their pull in a fixed sequence.

Actins↗