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H E Harris

Publications and source records attributed to H E Harris.

At least 19 recordsLinked to original sources
PubMed1988

The binary complex of pig plasma gelsolin with Mg2+-G-actin in ATP and ADP.

Pig plasma gelsolin combined with Mg-G-actin at less than 10(-8) M Ca2+ to yield a binary complex. Complexes formed from G-actin with bound ATP or ADP. They contained approx. 1 mol of non-exchangeable nucleotide per mol of actin. ATP hydrolysis was not coupled to binary complex formation, but ATP in the complex hydrolysed very slowly. The nucleotide in the binary complex behaved like one of the two nucleotide molecules in the ternary complex (two actin monomers to one gelsolin), but the actin-gelsolin interaction was weaker in the binary complex.

Actins↗
PubMed1986

Preparation and characterization of pig plasma and platelet gelsolins.

Pig plasma gelsolin has been prepared by a revised method involving poly(ethylene glycol) precipitation, chromatography on CM-cellulose and affinity chromatography on actin-Sepharose. Pig platelet gelsolin has been prepared by chromatography on DEAE-cellulose and actin-Sepharose. Partial chemical and proteolytic cleavage shows that the two proteins are closely related in their fragmentation patterns. The amino acid sequences are identical at the N-terminus of the platelet protein, but the plasma protein has an additional nine residues on the N-terminal side of the common sequence. Calcium binding studies show that the plasma protein has similar calcium binding properties to both macrophage and platelet gelsolins.

Amino Acid Sequence↗
PubMed1985

Covalent complexes formed between plasma gelsolin and actin with a zero-length cross-linking compound.

Actin and plasma gelsolin were covalently cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Two major intermolecularly linked products were identified on polyacrylamide gels. By use of 14C-labeled actin and 125I-labeled gelsolin, these were shown to be the 1:1 and 2:1 complexes of actin with gelsolin, respectively. The higher molecular weight complex predominated under all conditions tested including the presence and absence of Ca2+. In titration experiments in which actin at different concentrations was reacted with a fixed concentration of gelsolin, end points were obtained for the formation of both cross-linked species at about two actins per gelsolin, implying that a 2:1 noncovalent complex is cross-linked. In 0.1 mM Ca2+, the extent of cross-linking was independent of protein concentration down to 50 nM gelsolin. At low Ca2+ concentrations (less than 10(-8)M), the extent of cross-linking was very much reduced at micromolar gelsolin and fell to zero at about 100 nM gelsolin. The binding of actin to gelsolin to give a cross-linkable complex is therefore very strong at 0.1 mM Ca2+ but much weaker at low Ca2+ concentrations.

Actins↗
PubMed1985

Lack of nucleotide cleavage on the binding of G-actin-ATP to plasma gelsolin.

G-Actin-ATP bound to plasma gelsolin to form a 2:1 complex. The complex contained approximately equivalent amounts of nucleotide and actin. More than 84% of this nucleotide was ATP. Half of the bound nucleotide was displaced by cold chase and the remainder did not exchange, implying that the two actins in the complex are not equivalent.

Actins↗
PubMed1984

Plasma gelsolin caps and severs actin filaments.

Plasma gelsolin caps actin filaments at their 'barbed' ends and severs them along their length. Capping has been demonstrated both by direct visualization using gold-labeled gelsolin and by inhibition of actin polymerization onto the barbed ends of fragments of the acrosomal process of Limulus sperm. Severing activity is demonstrated by the fact that actin filaments nucleated off acrosomal fragments are shortened or removed within a few seconds by added plasma gelsolin without any obvious disruption of the actin bundles in the acrosomal processes themselves.

Acrosome↗
PubMed1983

Plasma actin depolymerizing factor has both calcium-dependent and calcium-independent effects on actin.

The effects of pig plasma actin depolymerizing factor (ADF) on both G-actin polymerization and F-actin fragmentation have been examined by using rabbit skeletal muscle actin labeled with N-(1-pyrenyl)iodoacetamide, a sensitive fluorescent probe for monomer to filament interconversion. Fluorescence data have been compared with results obtained by viscometry and by difference absorption measurements at 232 nm. Plasma ADF nucleates actin filament assembly in a Ca2+-dependent manner; actin polymerization rates are enhanced at greater than 10(-6) M Ca2+. The calcium concentration dependence of this effect, showing a shift in ADF nucleating capacity between 10(-6) and 10(-7) M Ca2+, is that expected for an intracellular regulatory effect, but in plasma, the protein would always be saturated with Ca2+. Although the rate of polymerization is markedly enhanced in the presence of calcium ions, the extent of polymerization (as determined by the amplitude of the fluorescence change or the specific viscosity) is reduced in the presence of ADF and shows little or no Ca2+ dependence. The critical concentration of actin monomers is increased in the presence of ADF whether calcium is present or not. When ADF is added to F-actin, there is an immediate fall in fluorescence. This conversion of filaments to monomers by ADF (as defined by the fluorescence changes) is unaffected by calcium concentration. Electron micrographs of F-actin treated with ADF show that the filaments are indeed shortened at both high and low calcium concentrations. Taken together, these observations are interpreted in terms of a model in which ADF has both Ca2+-sensitive and Ca2+-insensitive binding sites for actin.

Actin Depolymerizing Factors↗
PubMed1977

Actin from the nematode, Caenorhabditis elegans, is a single electrofocusing species.

We have prepared actin from wild type Caenorhabditis elegans animals by three procedures: a purification dependent on the ability of actin to form F-actin, affinity chromatography which preferentially binds G-actin, and co-precipitation of an actin-myosin complex by antimyosin antibodies. Each preparation yields a single electrofucsing species of actin. Comparison of actin from C. elegans embryos and animals reveals that embryos also have the same single electrofocusing species of actin.

Actins↗
PubMed1977

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.

Actins↗
PubMed1977

Myosin and paramyosin of Caenorhabditis elegans: biochemical and structural properties of wild-type and mutant proteins.

Myosin and paramyosin have been purified from the nematode, Caenorhabditis elegans. The properties of the myosin in general resemble those of other myosins. The native molecule is a dimer of heavy (210,000 dalton) polypeptide chains and contains 18,000 and 16,000 dalton light chains. When rapidly precipitated from solution, it forms small, bipolar aggregates, about 150 nm long, consistent with the expected molecular structure of a rigid rod with a globular head region at one end. Its ATPase activity is stimulated by Ca2+ and EDTA. The myosin binds to F actin in a polar and ATP-sensitive manner, and the Mg2+-ATPase is activated by either F actin or nematode thin filaments. Dialysis of myosin to low ionic strength produces very long filaments. When a myosin-paramyosin mixture is dialyzed under the same condtions, co-filaments form which consist of a myosin cortex, surrounding a paramyosin core. Some properties of myosin from the mutants E675 and E190, which have functionally and structurally altered body wall muscles, are compared with those of wild-type myosin. These myosins of these results are discussed in terms of the myosin heavy chain composition.

Actins↗
PubMed1976

Myosin-paramyosin cofilaments: enzymatic interactions with F-actin.

The interaction between paramyosin and myosin has been studied by enzymological methods. Clam adductor paramyosin inhibits the actin-activated, Mg2+-requiring ATPase of both clam adductor and rabbit skeletal muscle myosins. Myosin and paramyosin must be rapidly coprecipitated for this inhibition. Incubation with F-actin in the absence of ATP does not alter this effect. This inhibition follows a hyperbolic function with respect to paramyosin concentration. Slow precipitation by dialysis of myosin and paramyosin together leads to copolymers with actin-activated ATPase equivalent to that of slowly formed myosin filaments. Both kinds of slowly formed filaments have enzymatic properties distinct from those of the rapidly precipitated proteins. Paramyosin is competitive with F-actin for their effects upon myosin. The apparent affinity of myosin for F-actin is markedly reduced by association with paramyosin, but the extrapolated maximal velocity of actomyosin is unaffected. The specificity of this inhibition is strongly suggested by marked quantitative differences between native and cleaved paramyosins. No inhibition of intrinsic myosin ATPase by paramyosin is seen. These studies suggest that at least two types of condition-dependent association between myosin and paramyosin are possible. One class of interactions is associated with enzymic inhibition in rapidly coprecipitated filaments, whereas slowly formed cofilaments exhibit catalytic activity similar to that of identically treat-d myosin and have a characteristic 14.5 nm axial repeat.

Actins↗
PubMed1975

Conformational properties of pig-heart cytoplasmic aspartate aminotransferase. Circular-dichroism and absorption-spectroscopic study of dicarboxylate binding.

1. The interaction between aspartate aminotransferase and dicarboxylates of various chain lengths and geometries has been studied from pH 6.5 TO 8.5 by circular dichroism (CD) and absorption spectroscopy. Liganding causes protonation of the pyridoxal phosphate-enzyme Schiff's base complex; the consequent changes in optical properties deltaAlambda, deltaCDlambda at the coenzyme maxima (lambda = 363 or 430 nm) are analysed for binding constants and the degree of perturbation of the coenzyme protonic dissociation constant, pKa. 2. Aliphate dicarboxylates follow linear binding functions for all optical parameters; in contrast, m and p-phthalates follow non-linear binding functions for both deltaAlambda and deltaCDlambda, implying that successive phthalate ligands bind with decreasing affinity. The ratio detlaCDlambda is effectively constant for a given ligand and the characteristic values for aromatic ligands indicate a changed environment for the coenzyme. 3. Inspection of the non-linear process for phthalates suggests that initially, binding occurs with high affinity, but with characteristically small effects on pKa. It is inferred that alipathic and aromatic dicarboxylates bind at different subsites in the active site region, perturbing the coenzyme pKa by an indirect protein-mediated mechanism. 4. Non-linearity of binding could derive from multiple binding to an individual subunit. Alternatively, different single sites may exist on adjacent subunits of the dimer, implying non-equivalence between otherwise identical subunits, expressed in properties involving groups close to the active site.

Animals↗
PubMed1975

Interaction of myosin and paramyosin.

The interaction of myosin and paramyosin was investigated by enzymological and ultrastructural techniques. The actin-activated Mg+2 ATPase of rabbit skeletal muscle myosin can be inhibited by clam adductor paramyosin. Both proteins must be rapidly coprecipitated to form filaments for this inhibition. Slowly formed cofilaments are fully activatable by F-actin. In both cases, the cofilaments possess unique structural characteristics when compared to homofilaments. The mode of inhibition appears to be competitive when different concentrations of paramyosin and F-actin are compared. The apparent affinity of the myosin heads for actin is reduced by the presence of paramyosin within rapidly reconstituted thick filaments. These results suggest that paramyosin may serve as part of a relaxing mechanism within invertebrate muscles. It is unlikely that paramyosin plays a role in the initiation and maintenance of catch within specialized molluscan muscles.

Actins↗