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J F Marecek

Publications and source records attributed to J F Marecek.

32 records · Page 2Linked to original sources
PubMed1984

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria.

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.

Adenosine Triphosphatases↗
PubMed1983

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin.

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.

Actomyosin↗
PubMed1982

Inhibition of oxygen exchange by chemical modifiers at the sulfhydryl 1 or reactive lysine residue of myosin. Changing the rates of intermediate enzymatic reactions by modifying the course of reaction-linked conformational changes.

During the hydrolysis of MgATP by myosin, there is an extensive exchange of oxygen between water and the terminal phosphate group of bound nucleotide, which results from a repeated cycle of hydrolytic cleavage and its reversal. An analysis of the distribution of [18O]Pi species from the hydrolysis of [gamma-18O]ATP gives an estimate for the apparent rate constant [k-3(app)] of reverse cleavage, which is the rate-limiting step of the exchange cycle. We have performed this kind of analysis with two different forms of modified myosin, containing either N-ethylmaleimide at the sulfhydryl 1 group or trinitrophenyl at the reactive lysine residue. Although these modifications of the protein are chemically different, and the sulfhydryl 1 group and the reactive lysine residue are far apart in the primary chain of the myosin head, the two modifications caused a similar marked inhibition of oxygen exchange. This effect resulted from: 1) a decrease in the time available for the exchange cycle due to a 3-10-fold increase in the turnover rate of hydrolysis and 2) a reduction in k-3 (app) from 2-5 s-1 to 0.2-0.5 s-1. It is proposed that modifications of this type influence enzymatic activity by altering the course of "reaction-linked conformational changes," i.e. those changes in protein structure that are coupled to the catalytic mechanism but are not part of the substrate-binding site proper. It is also suggested that certain naturally occurring protein modifications, e.g. methylation of histidine or lysine residues, could regulate function by operation of this mechanism.

Adenosine Triphosphate↗
PubMed1982

Effects of borohydride-treated oligomycins on processes of energy transduction in mitochondria.

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7-keto and the 11-keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP-dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration-linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride-treated antibiotics are also observed in rat-liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4-dinitrophenol), than of Pi formation stimulated by certain amine-fluorescamine modifiers (e.g.) the benzylamine-fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler-stimulated and modifier-stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+-ATPase as a result of hydrogen bonding to, at least, the 7-keto and/or the 11-keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen-bonding is less efficient due to the pronounced directional characteristic of hydrogen-bonding to keto groups.

Adenosine Triphosphatases↗
PubMed1981

Inhibition of the links between electron transfer and proton translocation in mitochondria.

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine-specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine-containing antibiotic amphotericin B. The effect of these 'modifiers' (i.e. fluorescamine transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites II and III. In contrast, the lactone derivative (or cyclic form) of the amine-fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites II and III are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects; (c) specific structural features in the amine-fluorescamine compounds, and in the mitochondria-fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the 'redox loop' model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.

Animals↗
PubMed1980

Phosphatidylcholesterol bilayers. A model for phospholipid-cholesterol interaction.

Aqueous dispersions of monovalent and divalent cations salts of O-(1,2-dipalmitosyl-sn-glycero-3-phosphoryl)cholesterol form multilamellar vesicles as shown by freeze-fracture electron microscopy, by electron micrographs of the negatively stained liposomes, and by swelling curves of liposomes in hypo-osmotic medium. Differential scanning calorimetry reveals that aqueous dispersions of divalent metal salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol undergo a characteristic thermotropic phase transition with a relatively large cooperative unit (n greater than 250 for the calcium salt). In contrast, monovalent cation salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol do not show a thermotropic phase transition under comparable conditions. The molecular area of 0-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol in a monolayer is the same in the presence and absence of Ca2+, and is virtually equal to the area of an equimolar mixture of dipalmitoyl phosphatidic acid and cholesterol. To account for the novel state induced by Ca2+, on aqueous dispersions of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol (i.e., bilayer organization and highly cooperative phase transition), a linear array model is proposed in which Ca2+ bridges adjacent arrays of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol molecules, thus freezing the acyl chains in their normal state. One of the main corollaries of the model is that the cooperative unit for a thermotropic phase transition is essentially one-dimensional, rather than a two-dimensional matrix. O-(1,2-Dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol is proposed as an orientationally and conformationally restricted analog of glycerophospholipid plus cholesterol in bilayers.

Cholesterol↗
PubMed1980

Differential effects on energy transduction processes by fluorescamine derivatives in rat liver mitochondria.

Intact rat liver mitochondria were treated with compounds derived from the reaction of fluorescamine with various types of primary amines, including the mycosamine-containing antibiotics amphotericin B and nystatin. The effect of varying amounts of these compounds on ATPase-linked inorganic phosphate (Pi) formation on oxygen consumption, and on MgATP-linked and succinate-linked proton movements was examined. The antibiotic-fluorescamine compounds did not affect the Pi formation rate but strongly inhibited both the ATPase-linked and the succinate-linked H+ extrusion rates to approximately the same extent. The antibiotic derivatives decreased the oxygen consumption rate, but this effect was much smaller than the decrease in the respiration-dependent proton extrusion rate. The benzylamine-fluorescamine compound significantly increased the Pi formation rate, in contrast to the antibiotic analogues. The benzylamine derivative, like the antibiotic derivatives, inhibited both types of proton extrusion rates. The slight decrease in the oxygen consumption rate caused by the benzylamine derivative was significantly smaller than the corresponding decrease observed with the antibiotic derivatives. These studies, in which fluorescamine derivatives bind reversibly to mitochondria, are compared with previous studies in which fluorescamine itself binds irreversibly to mitochondria and results in a Pi formation rate increase and MgATP- and succinate-linked proton extrusion rate inhibition but has no effect on the oxygen consumption rate.

Adenosine Triphosphatases↗
PubMed1979

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects.

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.

Cardiolipins↗
PubMedDate not supplied

Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid.

New polyvalent hydrazide cross-linkers were synthesized, characterized, and used to prepare hydrazide cross-linked hydrogels derived from hyaluronic acid (HA). First, the chemical synthesis and characterization of the di-, tri-, tetra-, penta-, and hexahydrazides are presented. Second, HA concentration, buffer type and concentration, and ratio of HA to carbodiimide to cross-linker were varied to obtain HA-hydrogels with different chemical and physical properties. Third, two new assays are described to monitor the stability of HA-hydrogels toward hyaluronidase (HAse) and other media. These assays were used to evaluate the stability of cross-linked HA-hydrogels to HAse solutions and different pH values. Hydrophobic cross-linkers gave the most stable gels, and the susceptibility of the gels to HAse was independent of cross-linker concentration. HAse does not significantly penetrate the HA-hydrogels and acts primarily at the gel-solution interface. The HA-hydrogels are stable in acid environments and dissolve gradually above pH 7.0.

Biotransformation↗