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S R Anderson

Publications and source records attributed to S R Anderson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Flexibility involving the intermolecular dityrosyl cross-links of enzymatically polymerized calmodulin.

The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves range from 13 ns (zero Ca2+) to approximately 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the global rotations of polymer molecules are detected near the operating limits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 ns (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., and Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi2 = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) to 23% (+Ca2+) of the anisotropy.

Animals↗
PubMed1997

Identification of a new phosphorylation site in cardiac muscle phosphofructokinase.

The novel phosphorylation site (Ser376) that we discovered during in vitro studies of the troponin C- or calmodulin-induced phosphorylation of rabbit muscle phosphofructokinase [Zhao, Z., Malencik, D.A., and Anderson, S. R. (1991) Biochemistry 30, 2204] also undergoes phosphorylation in the epinephrine-stimulated rabbit heart. Reversed-phase HPLC and/or iron chelate affinity chromatography performed on CNBr digests of phosphofructokinase that had been isolated from epinephrine-treated hearts yields a largely phosphorylated peptide corresponding to amino acid residues 371-378 of the enzyme. Mass spectrometry, gas phase sequencing, and amino acid analyses establish the structure and phosphorylation state of the peptide.

Animals↗
PubMed1996

Dityrosine: preparation, isolation, and analysis.

This article describes chromatographic and spectroscopic techniques that have multiple applications in the preparation, isolation, and analysis of dityrosine. A three-step chromatographic procedure facilitates the preparation of 120 mg or more (yield > 26% of theoretical maximum) of dityrosine from the enzyme-catalyzed oxidation of tyrosine. DEAE-cellulose chromatography performed in a boric acid-sodium borate buffer removes most of the contaminating pigments. Two-dimensional pH-dependent chromatography on BioGel P-2 separates dityrosine from tyrosine, residual pigments, salts, etc. Elemental analysis indicates that the purified product is approximately 92% dityrosine by weight. Fast atom bombardment mass spectrometry and two types of reverse-phase high-performance liquid chromatography (HPLC), monitored in fluorescence and absorbance measurements, verify the purity of the dityrosine. The distinctive pH-dependent chromatography of dityrosine on BioGel P-2, with reversible adsorption to the matrix occurring at pH values less than 3, is useful for the isolation of varying quantities of dityrosine and for analysis per se. Affinity chromatography on immobilized phenyl boronate (Matrex Gel PBA-60) is an alternate method for the isolation and determination of dityrosine, which undergoes specific interactions with the boronate moiety and possible hydrophobic association with the phenyl group. Two new reverse-phase HPLC techniques expedite the analysis of picomole quantities of dityrosine. One employs isocratic elution (92% H2O, 8% acetonitrile, and 0.1% trifluoroacetic acid) of an ODS II Spherisorb column, with both fluorometric and spectrophotometric detection. The other procedure may be performed in conjunction with total amino acid analysis. A rapid gradient program, developed with a Phenomenex Ultracarb 20 column, clearly separates dabsylated dityrosine and tyrosine from other dabsylated amino acids. It is especially useful when dityrosine is a trace component.

Boronic Acids↗
PubMed1996

Rabbit liver phosphofructokinase: rapid purification and phosphorylation site identification.

Liver phosphofructokinase can be selectively precipitated by the addition of protamine sulfate to a heat-treated crude extract and redissolved, giving nearly full recovery of catalytic activity in combination with a 67-fold increase in specific activity. We have incorporated protamine sulfate precipitation into a five step purification procedure that can be completed in one day--giving 47% recovery of electrophoretically homogeneous liver phosphofructokinase having a specific activity of 50 units/mg. The radio-labeled fragment isolated from a CNBr digest of liver phosphofructokinase that has undergone in vitro phosphorylation catalyzed by the cAMP-dependent protein kinase has the sequence AEYVSGELEHVTRRSLS.

Adenosine Triphosphate↗
PubMed1996

Dityrosine formation in calmodulin: cross-linking and polymerization catalyzed by Arthromyces peroxidase.

We employ bovine brain calmodulin, a protein that is subject to photoactivated dityrosine formation [Malencik, D. A., & Anderson, S. R. (1987) Biochemistry 26, 695; (1994) Biochemistry 33, 13363], as a model for the development of an efficient enzyme-catalyzed protein cross-linking technique. Key steps in the elaboration of the procedure are (1) identification of a peroxidase, from Arthromyces ramosus, that catalyzes dityrosine production in proteins that are not acted on by other common peroxidases, (2) monitoring of the intrinsic fluorescence of dityrosine to determine optimum reaction conditions, achieved with calmodulin in solutions containing boric acid-sodium borate (concentration > or = 0.2 M), approximately pH 8.3, approximately 40 degrees C, and (3) quenching of the reaction with reduced glutathione. Arthromyces peroxidase is the only common peroxidase able to catalyze significant dityrosine production in calmodulin, through a reaction that is largely intermolecular. Gel filtration yields fractions (accounting for approximately 40% of the initial calmodulin) that represent differing mobility ranges in NaDodSO4 polyacrylamide gel electrophoresis and contain close to the maximum possible amounts of dityrosine. The various fractions undergo Ca2+-dependent conformational changes detected in sedimentation velocity and/or fluorescence anisotropy measurements. Most of the samples stimulate the Ca2+-dependent activity of smooth muscle myosin light chain kinase. In catalytic assays utilizing the synthetic phosphatic acceptor peptide, the average activities range from 50 to 100% of that determined for native calmodulin. However, only the least polymerized fraction and the photogenerated calmodulin dimers significantly enhance the p-nitrophenylphosphatase activity of calcineurin. The ability to prepare soluble calmodulin polymers that retain a substantial degree of biological activity and exhibit the intense visible fluorescence of dityrosine illustrates the potential usefulness of Arthromyces peroxidase in the zero-length cross-linking of proteins.

Animals↗
PubMed1994

Dityrosine formation in calmodulin: conditions for intermolecular cross-linking.

The pattern and extent of photoactivated dityrosine formation in bovine brain calmodulin are strongly affected by the presence of superoxide dismutase during UV irradiation. The addition of the enzyme to Ca(2+)-containing solutions of calmodulin results in an altered distribution of the dityrosine-containing photoproducts, from a predominance of cross-linked monomer to a mixture of products with inter- and intramolecular cross-linking. When Ca2+ is absent, significant dityrosine formation occurs only in the presence of superoxide dismutase. Fractionation of the latter reaction mixture yields a dimer of calmodulin, corresponding to a single component in sedimentation equilibrium, a smaller amount of a more highly polymerized material, and virtually no cross-linked monomer--as was found in the absence of the enzyme [Malencik, D.A., & Anderson, S.R. (1987) Biochemistry 26, 695]. Although it is homogeneous in terms of molecular weight, the purified dimer contains three electrophoretic components. Chemical characterization demonstrates intermolecular cross-linking of Tyr-99 to Tyr-138 and probably of Tyr-99 to Tyr-99. On the average, 85-90% of the dimeric calmodulin molecules bind two molecules of smooth muscle myosin light chain kinase. Catalytic activity determinations with this enzyme detect no difference between the dimer and the native protein. Fluorescence anisotropy measurements of Ca2+ binding give a Hill coefficient of < or = 0.73 and a free Ca2+ concentration of approximately 11 microM at 50% saturation. The average pKa of the dityrosine cross-link in the dimeric calmodulin is 8.5-8.6 (+/- Ca2+). Sedimentation velocity experiments show that the average Ca(2+)-liganded dimer has an elongated structure, with a relative frictional ratio approximately 30% greater than that of the native monomer. In terms of functional properties, the dimeric calmodulin preparation is more like the native protein than it is like the previously reported cross-linked monomer.

Amino Acids↗
PubMed1994

Primary and secondary kinetic isotope effects as probes of the mechanism of yeast enolase.

Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Kinetic isotope effects have been used to determine whether abstraction of the proton from C-2 and loss of hydroxide from C-3 of 2-phosphoglycerate occur in a concerted reaction or as sequential processes and whether these steps are kinetically significant for the enolase-catalyzed reaction. Enolase exhibits a significant primary deuterium isotope effect, as well as catalyzing the relatively rapid exchange of the C-2 proton with solvent water. Secondary C-3 deuterium isotope effects are also reported, both when the C-2 carbon carries a hydrogen and when this center is deuterated. These results provide information about the kinetic significance and timing of the transition state(s) associated with the loss of H+ and OH-. Strong evidence has been presented for a stepwise mechanism where both the rate of proton abstraction and one or both of the later transition states, i.e., those associated with hydroxide loss and product release, limit the overall reaction rate. If a concerted reaction were to be invoked, the presence of a small secondary 2H isotope effect in combination with the observed rate of exchange of the C-2 proton require the intrinsic secondary 2H kinetic isotope effect to be effectively unity. For the concerted mechanism, an intrinsic effect of unity would be consistent only with an extremely asymmetric transition state that is dominated by C-H bond cleavage.

Deuterium↗
PubMed1994

Racemization and oxidation studies of hair protein in the Homo tirolensis.

Amino acids contained in fossil materials show an increasing extent of racemization with age, postulating time and temperature as the two major variables. The recent discovery of the Homo tirolensis made possible a comparison between racemization rates of the amino acids found in hair at identical ages (5200 years of age) but at different diagenetic temperatures ("Ginger," found in the hot, dry sand of Egypt; H. tirolensis, found on a glacier of the Otztaler Alps). The rate of racemization was higher in the H. tirolensis, which is surprising and in contrast to current concepts. Ortho-tyrosine and di-tyrosine, parameters for OH-radical attack, were also higher in the H. tirolensis, suggesting a role for free OH-radical involvement in the racemization process.

Amino Acids↗
PubMed1993

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase.

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). This active preparation consists of three related species containing residues 1-290, 1-296, and 1-298 of the 44.7 kD gamma-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740-11745, 1991). Good recoveries of catalytic activity--with varying degrees of calcium dependence--result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein. Physical characterization shows that the 33 kD chymotryptic fragment is globular, with S20,w = 2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylase a and b, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344-350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the gamma-subunit of phosphorylase kinase.

Amino Acid Sequence↗
PubMed1992

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.

Hydrogen-Ion Concentration↗
PubMed1992

Development of a continuum of services for children and adults with autism and other severe behavior disorders.

The development of a 12-step continuum of services for individuals with autism is described. The operation and funding of outreach parent training; homebased early intervention; preschool, vocational, and adult intermediate care; and school consultation programs are outlined. The use and importance of evaluative data on both treatment outcome and staff skills are emphasized.

Activities of Daily Living↗
PubMed1992

Analytical sedimentation studies of turkey gizzard myosin light chain kinase and telokin.

Sedimentation equilibrium and velocity studies were performed with turkey gizzard myosin light chain kinase (MLCK) and telokin, a small protein apparently corresponding to the sequence of the COOH-terminal end of MLCK. The measurements carried out with MLCK give values for the monomer molecular weight (M(r)), sedimentation coefficient (S20 degrees,w), and virial coefficient (A2) of 108,000, 3.74 S, and -1.95 x 10(-4) mol.ml.g-2, respectively. In the case of telokin, M(r) = 18,500; S20 degrees, w = 1.63 S; and A2 = 5.81 x 10(-4)mol.ml.g-2. Combination of the results of the two kinds of experiment shows that MLCK is a rod-shaped molecule (a/b = 18.9) with a Stoke's radius of 69 A. Telokin is also elongated (a/b = 8.3) with a Stoke's radius of 29 A. MLCK apparently exhibits self-association, with 15% of the protein sedimenting as a dimer in the experiments.

Amino Acids↗
PubMed1991

Fluorometric characterization of dityrosine: complex formation with boric acid and borate ion.

Borate/boric acid solutions have distinctive effects on the absorption and fluorescence emission spectra of dityrosine. In the presence of excess borate/boric acid, the fluorescence emission maximum of the singly ionized dityrosine chromophore shifts from 407 nm (quantum yield = 0.80) to 374 nm (quantum yield = 0.14). Fluorescence measurements performed as a function of pH and concentration are consistent with a 1:1 complex which may dissociate to either boric acid and singly ionized dityrosine (K1 = 17 mM) or to monoborate ion and unionized dityrosine (K2 = 0.10 mM). As a consequence of the pKa values characteristic of dityrosine and boric acid, complex formation is maximal near pH 8. 2,2'-Dihydroxy-biphenyl shows similar interactions. The fluorescence of dityrosyl calmodulin (0 Ca2+) also responds to the addition of boric acid, giving K1 = 42 mM and K2 = 2 mM. Singly ionized dityrosine produced through dissociation occurring in the excited state does not interact with boric acid.

Borates↗
PubMed1991

Characterization of a new substrate for protein kinase C: assay by continuous fluorometric monitoring and high performance liquid chromatography.

A synthetic peptide derived from the phosphorylation site in the beta-subunit of phosphorylase kinase (RTKRSGSVYEPLKI) is an efficient substrate for rat brain protein kinase C: Km = 18 +/- 2 microM and Vmax = 2.1 +/- 0.1 mumol/min/mg. The phosphorylation of the peptide, which occurs at Ser7, can be followed by four independent procedures. 1. Standard measurement of 32P incorporation. 2. Reverse phase HPLC in a gradient system containing 0.1 M ammonium sulfate in the stationary phase. 3. Continuous fluorometric monitoring of the changes in intrinsic peptide fluorescence. 4. Continuous fluorometric determination of NADH oxidation in a coupled enzyme assay.

Amino Acid Sequence↗
PubMed1991

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.

Amino Acid Sequence↗
PubMed1991

Phosphorylase kinase: development of a continuous fluorometric assay for the determination of catalytic activity.

The preferential binding of 1-anilinonaphthalene-8-sulfonate by rabbit muscle phosphorylase a is the basis of a continuous fluorometric assay for phosphorylase kinase. The maximum rate of change in fluorescence (d delta F/dt) is dependent on both the concentration of phosphorylase kinase and on conditions, such as pH and calcium ion concentration, which affect the enzyme. Parallel measurements of the increases in fluorescence and of 32P incorporation demonstrate the existence of a distinct intermediate in the conversion of phosphorylase b to a. We have used the assay to monitor the increase in calcium-independent activity which accompanies the limited chymotryptic digestion of phosphorylase kinase.

Anilino Naphthalenesulfonates↗
PubMed1990

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.

Amino Acid Sequence↗