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E H Fischer

Publications and source records attributed to E H Fischer.

At least 19 recordsLinked to original sources
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

Tyrosine phosphatases and their possible interplay with tyrosine kinases.

Protein tyrosine phosphatases represent a new family of intracellular and receptor-linked enzymes. They are totally specific toward tyrosyl residues in proteins, and, with specific activities 10-1000-fold greater than those of the protein tyrosine kinases, they can be expected to tightly control the level of phosphotyrosine within the cell. Most transmembrane forms contain two conserved intracellular catalytic domains, as displayed by the leukocyte common antigen CD45, but highly variable external segments. Some are related to the neuronal cell adhesion molecules (NCAMs) or fasciclin II and others contain fibronectin III repeats; this suggests that these enzymes might be involved in cell-cell interaction. The intercellular enzymes appear to contain a highly conserved catalytic core linked to a regulatory segment. Deletion of the regulatory domain alters both substrate specificity and cellular localization. Likewise, overexpression of the full-length and truncated enzymes affects cell cycle progression and actin filament stability, respectively. The interplay between tyrosine kinases and phosphatases is considered. A hypothesis is presented suggesting that in some systems phosphatases might act synergistically with the kinases and elicit a physiological response, irrespective of the state of phosphorylation of the target protein.

Animals↗
PubMed1979

Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The sequences of two phosphopeptides isolated from the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase (type II) and from two of its cyanogen bromide fragments, have been determined. One phosphorylation site is a threonyl residue located approximately 180 residues from the blocked NH2 terminus. Its sequence is: -Gly-Arg-Thr-Trp-Thr(P)-Leu-Cys- and includes one of the three sulfhydryl groups present in the molecule. The second phosphorylated site within the sequence: -Val-Ser(P)-Ile-Asn- is located towards the carboxyl end of the protein where the other 2 cysteinyl residues also reside. The finding that phosphorylation of the catalytic subunit occurs on two discrete sites rather than at random suggests that it might be of physiological importance, e.g. in the regulation of enzyme activity.

Amino Acid Sequence↗
PubMed1979

Characterization of Ca2+- and Sr2+-activated tension in functionally skinned chicken fibers of normal and dystrophic skeletal and normal cardiac muscle.

The Ca2+ and Sr2+ activation of tension in functionally skinned chicken fibers of normal and dystrophic skeletal and normal cardiac muscle were studied. The muscles studied can be separated into two groups based upon their Ca2+ and Sr2+ sensitivities: those which are significantly more sensitive to Ca2+ than to Sr2+, pectoralis and posterior latissimus dorsi (PLD), and those which show no Ca2+/Sr2+ sensitivity difference, cardiac and anterior latissimus dorsi (ALD). This suggests that there is more than one type of Ca2+ site involved in Ca2+ control of muscle contraction in different muscle types and suggests that ALD and cardiac muscle may be controlled by a different type of binding site than PLD and pectoralis muscle. Dystrophic ALD and PLD muscles showed little change in their Ca2+ and Sr2+ sensitivities from those of normal muscles in contrast to the pectoralis which showed a decrease in both Ca2+ and Sr2+ sensitivity (approaching that of PLD) with the onset of dystrophy. Similarly, upon SDS polyacrylamide gel electrophoresis, dystrophic ALD and PLD muscles showed no difference in contractile proteins from those of normal muscles, in contrast to pectoralis muscle where the appearance of a 36,000 dalton protein band correlated with the onset of dystrophy and the changes in the Ca2+/Sr2+ activation properties of this muscle. The contractile protein band pattern of normal and dystrophic PLD and dystrophic pectoralis muscle were similar including the presence of the 36,000 dalton protein.

Animals↗
PubMed1979

Mode of stimulation by injection of cyclic AMP and external acidification of the sodium efflux in barnacle muscle fibres.

1. A study has been made in single barnacle muscle fibres of the effect of micro-injected pure protein kinase inhibitor (PKI) on the response of the Na efflux to injection of cyclic AMP and external acidification. 2. (i) Injection into fibres of 1.6 x 10(-4) M-pure PKI is without effect on the resting Na efflux. (ii) Injection of 1.6 x 10(4) M-pure PKI before 0.03 M-cyclic AMP causes a marked reduction in the magnitude of the response of the Na efflux to the nucleotide. The same is true when 10(-4) M-cyclic AMP is injected after PKI. (iii) Injection of partially pure catalytic subunits causes a sustained stimulation of the ouabain-insensitive Na efflux, which is almost completely reversed by injecting PKI. (iv) Injection of 100 mM-EGTA before PKI fails to alter the lowered response of the ouabain-insensitive Na efflux to injection of 10(-4) M-cyclic AMP. (v) Ouabain (10(-4) M) when applied following the injection of 10(-4) M-cyclic AMP causes a drastic fall in the stimulated Na efflux. 3. (i) Injection of 1.6 x 10(-4) M-pure PKI before or after external acidification fails to abolish or reduce the stimulatory response to acidification. (ii) Injection of 1.6 x 10(-4) M-pure PKI before acidification practically abolishes the response of the ouabain-insensitive Na efflux to 0.03 M-cyclic AMP in the presence of acidification. (iii) Radioimmunoassay of total cyclic AMP and cyclic GMP content in single fibres before and after acidification shows no appreciable alteration in nucleotide content following acidificiation. (iv) Injection of 100 mM-EGTA before acidification enhances the stimulatory response to acidification. (v) External application of Dantrolene (10(-5) M) fails to alter the size of the stimulatory response to acidification. 4. (i) Prior external application of 5 x 10(-4) M-benzolamide results in a marked reduction in the magnitude of the response of the ouabain-insensitive Na efflux to the injection of 3 x 10(-4) M-cyclic AMP. (ii) Benzolamide totally abolishes the response of the ouabain-insensitive Na efflux to the injection of catalytic subunits. 5. The evidence brought forward is compatible with the view that (a) The mechanism by which cyclic AMP stimulates the Na efflux involves activation by cyclic AMP of the cyclic AMP-dependent protein kinase system, and hence release of the catalytic subunit, and (b) the mechanism by which external acidification leads to stimulation of the Na efflux involves activation of a benzolamide-sensitive system, possibly carbonic anhydrase, rather than the adenyl cyclase system. The actions of cyclic AMP and catalytic subunits on the Na efflux are closely linked to activation of the benzolamide sensitive system.

Animals↗
PubMed1978

Sequence of the carboxyl-terminal 492 residues of rabbit muscle glycogen phosphorylase including the pyridoxal 5'-phosphate binding site.

This communication presents the strategy and experimental details which establish the amino acid sequence of the carboxyl-terminal 492 residues (residues 350 through 841) of rabbit muscle glycogen phosphorylase (EC 2.4.1.1). The heavy segment (Hs), derived from the native enzyme by limited proteolysis with subtilisin, was cleaved with cyanogen bromide to yield 15 fragments. The amino acid sequences of 12 of these are described herein. The sequence of 3 other fragments (CB17C, CB18, and CB15) is described in accompanying reports by Koide, A., et al., and Hermann, j., et al. ((1978) Biochemistry 17 (first and second papers, respectively, in a series in this issue)). These 15 fragments were aligned by analysis of three others generated by cleavage of the heavy segment Hs at asparaginylglycine bonds with hydroxylamine and of four more generated by acid cleavage of aspartylproline bonds. Lysine-679 was identified as the binding site of the essential cofactor pyridoxal 5'-phosphate. These data, together with those reported in the accompanying papers (vide supra), establish the complete sequence of the 841 amino acid residues in glycogen phosphorylase. They provide a chemical basis on which the relationship between structure and function of the enzyme can be examined.

Amino Acid Sequence↗
PubMed1977

Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinases.

The heat-stable protein inhibitor (Walsh, D. A., et al. (1971), J. Biol. Chem. 246, 1977--1985) of the cyclic adenosine 3',5'-monophosphate dependent protein kinase has been isolated in pure form from rabbit skeletal muscle after a 430 000-fold purification with a 47% yield. The four-step procedure involves sequentially a heat treatment, batchwise anion and cation exchange, and affinity chromatography on protein kinase catalytic subunit covalently coupled to Sepharose 4B. The inhibitor is an acidic protein (pI = 4.24) of molecular weight 11 300. It contains 98 amino acid residues none of which contains sulfur and only 2 (phenylalanine and tyrosine) are aromatic. The NH2-terminus is blocked. The muscle content is ca. 0.6 mg of inhibitor per L of intracellular water. The inhibitor is tightly bound to the catalytic subunit of protein kinase (Ki congruent to 2 X 10(-9) M) and acts competitively with respect to the protein substrates. Protein kinase recognizes a short stretch of the inhibitor sequence, in which arginyl side chains play a crucial role. A study of various competitive inhibitors of the kinase confirms the importance of guanidino groups and hydrophobic side chains in the specific interaction with the substrate binding site.

Amino Acids↗
PubMed1977

Comparative properties of vertebrate parvalbumins.

Pure parvalbumins isolated from turtle, chicken, and rabbit white skeletal muscle have been characterized in terms of their physical, chemical, and immunological properties. As for the parvalbumins of most fish and amphibians, they have sedimentation constants S20,w of approximately 1.45 +/- 0.25 S and molecular weights of approximately 12,000, with little or no evidence for aggregation. They contain no tryptophan, at most one tyrosine, and a high proportion of phenylalanine, resulting in characteristic absorption spectra. All three parvalbumins contain 2 g atoms of calcium/mol bound with a KDiss less than or equal to 10(-6) M. Complete removal of calcium can be achieved by treatment with EDTA and EGTA or by a purified preparation of fragmented sarcoplasmic reticulum. By a direct analytical procedure, the concentration of parvalbumins in white skeletal muscle from the turtle, chicken, and rabbit was estimated at approximately 9 to 11, 0.2 to 0.4, and 0.6 to 1.1 g/kg, respectively. No parvalbumin or immunologically cross-reacting material could be detected in chicken white breast muscle, and very little was found in rabbit red muscle. All three proteins are immunologically distinct. A "minor" isoparvalbumin (approximately 2% of the major component) was found in turtle muscle only.

Amino Acids↗
PubMed1977

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.

Amino Acids↗