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M Fry

Publications and source records attributed to M Fry.

At least 91 records · Page 5Linked to original sources
PubMed1981

Studies on the resolution of cytochrome oxidase.

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I-IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V-VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of heme a3. It is suggested that the fraction of subunits I-IV,VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.

Acetates↗
PubMed1980

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta.

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.

Animals↗
PubMed1980

The localization of tightly bound cardiolipin in cytochrome oxidase.

One to two molecules of tightly bound cardiolipin are associated with resolved fractions of cytochrome oxidase containing subunits I to III or I to IV. Large scale isolation of subunits I to IV indicates the presence of approximately 0.5 molecule of cardiolipin per molecule of subunit I. Lipoprotein staining of sodium dodecyl sulfate/urea/acrylamide gels of cytochrome oxidase support the findings that subunit I is a lipoprotein. The resistance of this tightly bound cardiolipin to organic solvent extraction suggests a specific association of some tenacity with the protein.

Binding Sites↗
PubMed1980

Phospholipids as the molecular instruments of ion and solute transport in biological membranes.

Partition studies have established that phospholipids generally have the capabilities to mediate the transmembrane transport of the full range of ions and solutes that physiologically cross biological membranes. The list of transportable species includes cations, anions, amino acids, citric acid cycle intermediates, nucleotides, and sugars. Phospholipid-mediated transport can be readily modulated by altering the phospholipid mixture or by addition of detergents, nucleotides, divalent metals, proteins, peptides, or ring compounds. Containment of phospholipid within channels in protein appears to be the precondition for the formation of the micellar structure requisite for solute transport. Phospholipid-mediated transport is postulated to be a central feature of energy coupling, membrane-spanning systems, and membrane-bound, phospholipid-requiring enzymes.

Biological Transport↗
PubMed1980

Relation between enzymic catalysis and energy coupling.

The principles that underlie enzyme catalysis also apply to energy coupling processes. A comparison is made between a kinase system that mediates the phosphorylation of glucose by ATP (hexokinase), as the prototype for enzymic catalysis, and the mitochondrial electron-transfer complexes, as the prototypes for energy coupling systems. Induced polarization of chemical bonds and charge separation and elimination are common component events of both enzyme catalysis and energy coupling. Thus, definite limits can be imposed on models of energy coupling; they must comply with the basic principles of enzymic catalysis.

Binding Sites↗
PubMed1980

Ion-transport chain of cytochrome oxidase: the two chain-direct coupling principle of energy coupling.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) couples the aerobic oxidation of ferrocytochrome c to the cyclical transport of monovalent cations or to the active transport of monovalent and divalent cations. This transport capability is mediated by an intracomplex ion-transport chain of two protein-bound molecules of cardiolipin per molecule of cytochrome oxidase. Cardiolipin in a two-phase system shows the identical ionophoric pattern as does the cytochrome oxidase coupled system. A molecular model of the cardiolipin chain suggests the possibility of a cage-like structure through which cations can be transferred from phosphate group to phosphate group. The ion-transport chain and the electron-transport chain are anchored to the same set of subunits (I+IV); the close proximity of the two chains argues for the direct coupling of electron and cation flow. The ion-transport chain of cytochrome oxidase provides an introduction to the molecular mechanisms by which ions are moved across membranes in energy-coupling systems.

Amino Acids↗
PubMed1980

On reagents that convert cytochrome oxidase from an inactive to an active coupling state.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of beef heart mitochondria, prepared by a standard method and brought to the highest purity level, is essentially inactive when tested in the aerobic assay involving oxidation of reduced cytochrome c by molecular oxygen. Three reagents (lysolecithin, Tween 20, and exogenous phospholipids) can convert cytochrome oxidase from an inactive to an active coupling state. These conversions are reversible: i.e., removal of the inducing agent leads to loss of activity. The evidence for the intrinsic coupling capability is that cytochrome oxidase in the active state invariably generates a proton gradient during respiration, and such gradient formation is demonstrable even when cytochrome oxidase is not inserted into a liposome.

Animals↗
PubMed1979

Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts.

About 30% of the proteins of adherent cultured chick embryo fibroblasts are not solubilized by the non-ionic detergent Triton X-100 and remain firmly attached to the substratum. The insoluble residue contains a considerable part of the cell's cytoskeleton and its major constituents are large external transformation-sensitive (LETS) protein, the heavy chain of myosin, a 52,000 molecular weight protein and actin. Kinetic studies reveal that cytoskeleton insolubility in Triton is acquired either concurrently with cell adhesion or very closely with it. Neither cell adhesion nor binding of the Triton cytoskeleton to the substratum require de novo synthesis of protein. In the attempt to assess the role of LETS protein in cytoskeleton attachment, we find that trypsin-detached cells rapidly acquire Triton-insoluble cytoskeleton although their LETS protein content is about 15--20% of its level in long-term cultures. Removal of the great majority of LETS molecules of adherent cultures by either urea or trypsin treatment does not affect the relative amount or composition of the anchored cytoskeletal proteins. Also, LETS protein of cultures exposed to cycloheximide for extended periods of time, is reduced to 10% of its maximum amount without much affecting the attachment and composition of the cytoskeleton. It is deduced that the great majority of LETS protein is not required for the attachment of the Triton cytoskeleton to the substratum.

Actins↗
PubMed1979

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells.

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.

Age Factors↗
PubMed1979

The effect of cephaloridine on the stability of rat kidney lysosomes.

The administration of cephaloridine to rats caused a decrease in the excretion of acid phosphatase into the urine. The antibiotic itself had no effect on urinary acid phosphatase and inhibitors or proteolytic enzymes were not present in the urine from treated rats. Cephaloridine may therefore be stabilizing the lysosomal membrane in vivo and experiments with isolated lysosomes confirm this hypothesis. The lysosomal integrity was followed by measuring the acid phosphatase activity and the light scattering properties of the particles. A good correlation was obtained between these parameters in the case of thermal disruption and progesterone induced lysis of the lysosomes and low concentrations of cephaloridine (0.1-1.0 mmol/1) protected the lysosomes against this form of damage.

Acid Phosphatase↗
PubMed1979

The monitoring of lysosomal integrity by pH-stat and light scattering measurements.

Light scattering measurements were used to monitor the integrity of isolated rat kidney lysosomes during prolonged incubation at 37 degrees C or following the addition of lysolecithin. The fall in extinction at 520 nm (E520) was shown to correlate very well with the fall in the particulate enzyme activity and the corresponding rise in the soluble enzyme activity. Measurements were also made of the release of H+ from the lysosomes into the suspending medium following treatment with lysolecithin. A good relationship was obtained between acidification of the medium and changes in the light scattering (E520) of the lysosomal suspension. The value of these techniques in following rapid changes in the integrity of lysosomes is discussed.

Animals↗