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Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 343 records · Page 19Linked to original sources
PubMed1984

Identification of the residues on cyclic GMP-dependent protein kinase that are autophosphorylated in the presence of cyclic AMP and cyclic GMP.

Autophosphorylation of cyclic GMP-dependent protein kinase (GMP:protein phosphotransferase, EC 2.7.1.37) in the presence of cyclic AMP and Mg-ATP has already been shown to result in the incorporation of up to 2.6 mol phosphate per mol subunit and decrease the A0.5 for cyclic AMP approx. 10-fold. The major sites of autophosphorylation have now been identified as serine-50, threonine-58, serine-72 and threonine-84. Serine-1 and serine-64 are phosphorylated to a minor extent. Threonine-58, which is initially phosphorylated most rapidly, is also the major site that is phosphorylated in the presence of cyclic GMP and Mg-ATP. Since autophosphorylation in the presence of cyclic GMP does not decrease the A0.5 for cyclic AMP, phosphorylation of serine-50, serine-72, or threonine-84 must be responsible for this effect.

Amino Acid Sequence↗
PubMed1984

Calmodulin antagonists inhibit activity of myosin light-chain kinase independent of calmodulin.

The calmodulin antagonists W-7, trifluoperazine and R24571 in vitro inhibited calmodulin-dependent and independent myosin light chain kinase activity with IC50 values of about 300 microM, 140 microM and 18 microM in the presence of 8 mg/ml myosin light chains. These IC50 values decreased to 15 microM, 6 microM and 2.5 microM when the concentration of myosin light chains was decreased to 0.4 mg/ml in the presence of saturating concentrations of calmodulin. Endogeneous tyrosine fluorescence of myosin light chain measured at 334 nm was quenched concentration dependently by trifluoperazine and R24571. In addition, fluorescence of W-7 measured at 370 nm was quenched concentration dependently by myosin light chains. The quenching of fluorescence which was independent of calcium, suggested that all three compounds bound to myosin light chain. The IC50 values for trifluoperazine obtained from fluorescence quench curves at different concentrations of myosin light chain were almost identical with those obtained under similar conditions from inhibition curves of myosin light chain kinase. These results indicate that 'calmodulin antagonists' inhibit the activity of myosin light chain kinase independent of calmodulin by binding to myosin light chain. The implication of this finding for the interpretation of results obtained in vivo by the use of 'calmodulin antagonists' is discussed.

Animals↗
PubMed1984

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2.

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.

Animals↗
PubMed1984

Tension transients in skinned muscle fibres of insect flight muscle and mammalian cardiac muscle: effect of substrate concentration and treatment with myosin light chain kinase.

Glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus were isometrically contracted in MgATP-salines (10 microM Ca2+; 1.5 mM Mg2+; pH 6.7; 22 degrees C and 20 mM PEP; 100 U/ml pyruvate kinase). The ratio of ATPase activity to tension decreased by a factor of 2 after reducing the ATP-concentration from 15 to 0.5 mM. At all ATP-concentrations (0.5-15 mM), the fibres showed tension adjustments in response to small step changes in length characteristic to an actively contracting muscle: i) an elastic phase which did not depend on ATP-concentration ii) a quick phase of stress relaxation with at least two exponential components; iii) a phase of delayed tension generation. An increase in size of the length step and/or a decrease of ATP-concentration slowed the quick phase and the delayed phase. Similar results have been obtained with skinned cardiac muscle (pig right ventricle). To see, how the isolated contractile system is affected by an increase in the light chain phosphorylation, tension transients were studied in skinned right ventricular muscle fibres before and after incubation with ATP gamma S (2 mM), pure myosin light chain kinase (9 micrograms/ml), Calmodulin (1 microM) and Ca2+ (0.8 microM). While isometric tension development elicited by 20 microM Ca2+ in the ATP salt solution was barely affected in presence of the enzyme, the ATPase activity was decreased by about 25% of the control. There was also a marked decrease (about 50%) in the contraction velocity as determined by the recovery of tension following a quick release. Quick stretches cause an immediate increase in tension followed by a rapid fall and a subsequent rise in tension. The velocity of this tension rise decreased by approximately 30% after incubation with myosin light chain kinase.

Adenosine Triphosphate↗
PubMed1984

cGMP and cAMP inhibit tension development in skinned coronary arteries.

The effects of physiological concentrations of cGMP and cAMP on tension development in skinned coronary arteries (Triton X-100) were studied. cGMP inhibited tension elicited at intermediate Ca2+ concentrations at pH 7.0 but not at more acidic or alkaline pH values. cAMP, on the other hand, decreased submaximal tension development independent of pH (from pH 6.5 to pH 7.2). Neither nucleotide affected tension development at maximally activating Ca2+ concentrations.

Animals↗
PubMed1983

Autophosphorylation of cGMP-dependent protein kinase is stimulated only by occupancy of one of the two cGMP binding sites.

cGMP-Dependent protein kinase contains, per subunit, 2 binding sites for cGMP. The apparent KD values for site 1 and 2 were 12 and 55 nM. The analogues 8-benzyl-amino-cAMP and N2-monobutyryl-cGMP bind preferentially to site 1 and 2, respectively. Both analogues stimulate autophosphorylation of the enzyme at concentrations at which only half of the phosphotransferase activity of the enzyme is expressed. Complete expression of the phosphotransferase activity requires a high concentration of each analogue and is accompanied by inhibition of the autophosphorylation reactions. It is concluded that occupancy of site 1 or 2 stimulates autophosphorylation while occupancy of both sites prevents autophosphorylation.

Adenosine Triphosphate↗
PubMed1983

Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake.

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.

Adenosine Triphosphate↗
PubMed1983

Cyclic AMP-dependent protein kinase does not phosphorylate cyclic GMP-dependent protein kinase in vitro.

The autophosphorylation reaction of purified cGMP-dependent protein kinase has been studied. Apparent initial rates of autophosphorylation in the absence of cyclic nucleotides and in the presence of cGMP and cAMP are 0.006, 0.04, 0.4 mol Pi incorp./min-1. mol cGMP-kinase subunit-1. In the presence of cGMP and cAMP approximately 1 and 2 mol Pi are incorporated/mol enzyme subunit. These values are independent of the enzyme concentration. Stimulation of autophosphorylation by cAMP is not due to activation of a contaminating cAMP-dependent protein kinase since: (a) addition of the heatstable inhibitor protein of cAMP-kinase does not inhibit autophosphorylation; and (b) catalytic subunit of cAMP-kinase added at a 10-fold excess over cGMP-kinase does not phosphorylate cGMP-kinase.

Animals↗
PubMed1983

Injection of catalytic subunit of cAMP-dependent protein kinase into isolated cardiac myocytes.

Cyclic adenosine 3',5'-monophosphate (cAMP) or the free catalytic subunit (C) of the cAMP-dependent protein kinase were pressure injected into single guinea pig ventricular cells. The following results were obtained: Injection of cAMP prolonged the action potential and shifted the action potential plateau to a more positive level. Under voltage clamp, cAMP injection increased the amplitude of the slow inward calcium current (Isi). Injection of C permanently prolonged the action potential and enhanced the amplitude of Isi by a factor of 2-4, depending on the amount of injected C. In the current-voltage relations the potential of maximum Isi and the apparent current reversal did not change. After maximum prolongation of the action potential due to repeated injections of C, even high concentrations of adrenaline did not further change the configuration of the action potential. In many experiments transient depolarizations appeared after the injection. Correspondingly, under voltage clamp transient inward currents occurred. C injection increased both the time-dependent and time-independent potassium outward current. In response to injection of the catalytic subunit, the isotonic contraction was larger in amplitude and relaxation was faster. It is concluded that the cAMP-dependent protein kinase increases the slow inward calcium current in the heart, presumably by phosphorylation of some membrane proteins.

Action Potentials↗
PubMed1983

Effect of calcium-antagonist and calmodulin-antagonist drugs on calmodulin-dependent contractions of chemically skinned vascular smooth muscle from rabbit renal arteries.

1. Renal arteries from rabbits were chemically skinned by incubation with Triton X-100, and subsequently stored in buffered glycerol. 2. In the presence of Mg-ATP, of EGTA-buffered calcium, and of calmodulin, miniature strips of the skinned arteries developed tension the strength of which was approx. 15-20% of that of viable renal arteries. 3. Tension development was dependent on the concentration of both calcium and calmodulin. 4. The effect of eight vasodilator drugs, the majority of them being "calmodulin antagonists" or "calcium antagonists", on the skinned arteries was assessed. In concentrations up to 10(-3) M, verapamil, D-600, and hydralazine proved to be ineffective, and the same was found with the dihydropyridine derivatives, nifedipine and felodipine, at 0.6 X 10(-3) M and 0.8 X 10(-4) M, respectively, i.e. at saturation in a 9:1 contracting buffer/ethanol mixture (v/v). 5. In a concentration-dependent manner, trifluoperazine, W-7, and fendiline relaxed Ca-calmodulin-induced tension or prevented tension development when given prior to the activation by Ca-calmodulin. However, considerably higher concentrations of the drugs were necessary for half-maximal relaxation than the reported concentrations for half-maximal saturation of hydrophobic binding sites at the calmodulin molecule. 6. These findings suggest that at therapeutic blood levels, the vasodilator properties of calcium antagonists and other direct vasodilators cannot be explained by interference with the binding of myosin light chain kinase to calmodulin.

Animals↗
PubMed1983

A simple solid phase enzyme immunoassay for aldosterone in plasma and saliva.

A sensitive, solid phase enzyme immunoassay for the determination of aldosterone in plasma and saliva has been developed. For this purpose anti-aldosterone coated tubes were used. The enzyme marker was a covalently linked aldosterone/horseradish peroxidase conjugate. The assay had a limit of detection of 5 pg/assay tube. The test requires neither centrifuge nor gamma or beta counter, but only a simple photometer. The free/bound separation is based on the solid phase technique with a simple washing step. The normal values for aldosterone in plasma and saliva before and after ACTH application were determined. Saliva, being easily collected by a non-invasive, stress-free technique, is an alternative to plasma. The aldosterone levels in patients with Conn's syndrome were significantly increased in plasma and also in saliva.

Addison Disease↗
PubMed1983

Identification of voltage operated calcium channels by binding studies: differentiation of subclasses of calcium antagonist drugs with 3H-nimodipine radioligand binding.

3H-Nimodipine (3H-NIM) is a high affinity radioligand suitable to study Ca2+ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (KD) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37 degrees C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of 3H-NIM can be removed by EDTA (EC50 20 microM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. Ca2+ -antagonist drugs can be divided into three main classes based on their interaction with the 3H-NIM binding site: Class I has one site law of mass action-displacement isotherms with 3H-NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca2+ -channel. Diltiazem is a Class III Ca2+ -antagonist. Our in vitro studies lead us to conclude that the Ca2+ -channel contains multiple regulatory sites at which drugs can act.

Animals↗
PubMed1982

cGMP-dependent protein kinase decreases calcium sensitivity of skinned cardiac fibers.

Chemically skinned (Lubrol WX) cardiac muscle fibers produce half-maximum isometric tension at pCa 6.18 (pH 6.7) in presence of MgATP (10 mM). After addition of cGMP (5 microM) and cGMP-dependent protein kinase (0.1 microM), the pCa required for half-maximum activation is 5.96, while maximum tension is not affected. Similar shifts in the tension/pCa-relationship have been observed after incubation of skinned cardiac muscle fibers with cAMP of catalytic subunit of the cAMP-dependent protein kinase. The shift in the Ca2+-sensitivity is associated with an increased incorporation of radioactivity into a Mr 28000 band (presumably troponin-I) and a Mr 145000 band.

1-Methyl-3-isobutylxanthine↗