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E H Fischer

Publications and source records attributed to E H Fischer.

At least 109 records · Page 6Linked to original sources
PubMed1982

Binding of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] by the regulatory subunits of adenosine cyclic 3',5'-phosphate dependent protein kinase.

Binding to the regulatory subunits of types I and II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase (RI and RII, respectively) produces large distinctive increases in fluorescence and optical activity of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] [bis(ANS)]. Both specific and nonspecific interactions are involved. Association of the regulatory subunits with either the catalytic subunit or cAMP results in dissociation of a major portion of the bound bis(ANS) as detected by changes in fluorescence and circular dichroism. The results are consistent with the accepted cAMP binding properties of RI and RII, showing cooperativity in case of RI and two heterologous binding sites for RII. cGMP has the same overall effect on bis(ANS) binding as cAMP. However, very high concentrations are required for complete dissociation of bis(ANS) from RII, consistent with the observation that cGMP is inefficient in bringing about the dissociation of the type II holoenzyme. Magnesium binding to sites having dissociation constants of ca. 12 mM increases the interaction of bis(ANS) with both of the isolated regulatory subunits. Experiments involving the 37 000-dalton fragment of RII indicate that the limited proteolytic cleavage was heterogeneous, with only 24-39% of the resulting population interacting strongly with the catalytic subunit.

Anilino Naphthalenesulfonates↗
PubMed1982

Complete amino acid sequence of human brain calmodulin.

The complete amino acid sequence of calmodulin from human brain has been determined by using peptides derived from digests with trypsin. Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides were purified by means of reversed-phase high-performance liquid chromatography and analyzed with a sequenator. The protein contains 148 amino acid residues and has a molecular weight of 16792. As in other calmodulins, the amino-terminal residue of the protein is blocked by an acetyl group, and a trimethyllysine residue is located at position 115. The only difference between this sequence and those fully determined in other species is the assignment of amide groups.

Amino Acid Sequence↗
PubMed1982

Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks.

A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.

Animals↗
PubMed1982

The influence of injected cyclic AMP protein kinase catalytic subunit on the sodium efflux in barnacle muscle fibres.

1. A study has been made of the response of the Na efflux in single barnacle muscle fibres to the injection of cyclic AMP protein kinase catalytic subunit (CSU).2. (i) Injected CSU stimulates the Na efflux in unpoisoned and ouabain-poisoned fibres. (ii) Stimulation in both situations is always sustained, and (iii) stimulation of the ouabain-insensitive Na efflux is observed with as low a concentration as 10(-7) M-CSU.3. (i) The response to injected CSU depends on [Ca](o) but is unaffected by prior external application of verapamil. (ii) An effect fails to occur in fibres cooled to 0 degrees C.4. (i) Injection of 5'-guanylylimidodiphosphate (Gpp(NH)p) following peak stimulation by CSU causes a further sustained stimulatory response. (ii) Injection of CSU following peak stimulation by Gpp(NH)p causes a further sustained stimulatory response. (iii) Injection of undialysed cholera toxin following CSU causes little or no effect, whereas injection of dialysed cholera toxin causes a stimulatory effect. Injection of CSU following undialysed cholera toxin causes little or no effect, whereas its injection after dialysed cholera toxin causes a fairly appreciable effect.5. (i) The response to CSU is reduced by prior injection of protein kinase type 2 regulatory subunit (R(II)) but not when R(II) is injected following peak response to CSU. (ii) Injection of protein kinase inhibitor partially reverses the response to CSU.6. (i) Injection of Mg(2+) fully reverses the response to CSU. (ii) Fe and Zn cause only partial reversal.7. (i) External imipramine but not trifluoperazine reduces the response to CSU. (ii) Diphenylhydantoin is ineffective.8. These results support the view that CSU behaves as a modulator of the ouabain-insensitive Na efflux and that Mg(2+), Fe and Zn are inhibitors of the catalytic reaction.

Animals↗
PubMed1981

Human brain calmodulin: isolation, characterization, and sequence of a half-molecule fragment.

A Ca2+-binding protein from human brain has been purified to homogeneity and identified as residues 72-148 of calmodulin. This half-molecule fragment (CaM72-148) contains 11 of calmodulin's 15 basic amino acids (including one trimethyllysine) and demonstrates a higher isoelectric point. Both tyrosines and three of eight phenylalanine residues also occur in the fragment, giving rise to a somewhat different absorption spectrum. Though it contains two of calmodulin's Ca2+-binding sites, CaM72-148 binds only one Ca2+ per molecule with a dissociation constant of 17 microM. No biological activity, as judged by its inability to activate cyclic nucleotide phosphodiesterase, is observed. The sequence of amino acids is identical with that of residues 72-148 of bovine brain calmodulin [Kasai, H., Kato, Y., Isobe, T., Kawasaki, H., & Okuyama, T. (1980) Biomed. Res. 1, 248-264]. CaM72-148 is thought to arise through proteolysis, and its implications for the structure and physiological role of calmodulin are discussed.

Amino Acid Sequence↗
PubMed1981

[Meiotic maturation of the ovocyte of Xenopus laevis : a 4-step mechanism].

Microinjection of the pure inhibitor (1) of cAMP dependent protein kinases (1.5 muM) into Xenopus oocytes triggered 100% germinal vesicle break down (GVBD), faster than progesterone and slower than the maturation promoting factor (MPF). Cholera toxin does not block (I) induced maturation; however cycloheximide inhibits (I) but not MPF induced GVBD.

Animals↗
PubMed1981

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation. A 4-step scheme for meiotic maturation.

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.

Animals↗
PubMed1981

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.

Amino Acid Sequence↗
PubMed1981

Rapid disassembly of cold-stable microtubules by calmodulin.

Purified cold-stable microtubules from the rat brain are insensitive to podophyllotoxin and to millimolar concentrations of free calcium. However, in the presence of calmodulin at concentrations substoichiometric to that of tubulin, calcium causes rapid microtubule disassembly. The half-maximal effective calcium concentration in the presence of calmodulin is 100 microM. With 800 microM free calcium, the half-maximal effective concentration of calmodulin is 1.0 microM (or one-tenth the tubulin concentration). Calmodulin is without effect in the absence of calcium. Troponin C is approximately one-fifth as effective as calmodulin, and parvalbumin is totally ineffective. Troponin I partially inhibits the calcium/calmodulin-induced disassembly of microtubules in the crude extract and blocks the calcium/calmodulin effect on purified cold-stable microtubules. A 5-fold excess of trifluoperazine does not inhibit the calcium/calmodulin-induced disassembly.

Animals↗
PubMed1980

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions.

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.

Animals↗
PubMed1980

Ca2+ and Sr2+ activation: comparison of cardiac and skeletal muscle contraction models.

The mechanism of contraction in rabbit fast-twich, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponin-tropomyosin regulated actin heavy meromyosin. The Ca2+ and Sr2+ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca2+ or Sr2+. By contrast, rabbit adductor magnus (fast-twich) skinned fibers required substantially higher [Sr2+] than [Ca2+] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr2+ than Ca2+ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca2+ and Sr2+ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca2+- and Sr2+-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.

Adenosine Triphosphatases↗