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F Hofmann

Publications and source records attributed to F Hofmann.

At least 289 records · Page 16Linked to original sources
PubMed1989

Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle.

Complementary DNAs for the beta subunit of the dihydropyridine-sensitive calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the beta subunit being a peripheral membrane protein associated with the cytoplasmic aspect of the sarcolemma. The protein contains sites that might be expected to be preferentially phosphorylated by protein kinase C and guanosine 3',5'-monophosphate-dependent protein kinase. A messenger RNA for this protein appears to be expressed in brain.

Amino Acid Sequence↗
PubMed1989

The cDNA of the two isoforms of bovine cGMP-dependent protein kinase.

cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.

Amino Acid Sequence↗
PubMed1989

The amino terminus regulates binding to and activation of cGMP-dependent protein kinase.

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.

Adenosine Triphosphate↗
PubMed1989

Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Decreased cardiac concentration of cGMP kinase in hypertensive animals. An index for cardiac vascularization?

Cyclic GMP (cGMP) kinase is intimately involved in the regulation of vascular smooth muscle tone. Its tissue concentration was determined in normotensive and hypertensive rats by use of monospecific anti-cGMP kinase antibodies. Hearts of spontaneously hypertensive rats and renovascular (Goldblatt II) hypertensive rats contained half the concentration of cGMP kinase than those of the respective normotensive animals. The increase in blood pressure and the resulting left ventricular hypertrophy were correlated inversely with the left ventricular cGMP kinase concentration. This decrease was specific for the left ventricle and was not observed in other tissues. In addition, the cardiac concentration of cGMP kinase was unchanged in hyperthyroid animals that had comparable left ventricular hypertrophy and mild hypertension. This suggested that in severe renovascular hypertension the decrease in cardiac cGMP kinase concentration is caused by a relative lack of cardiac vessel growth during the development of hypertrophy. In agreement with this conclusion, immunohistochemistry of cardiac cross sections showed that cGMP kinase was exclusively located in cardiac vessels. In support of this localization, the maximal arterial blood flow of heart, liver, skeletal muscle, and kidney correlated excellently with the cGMP kinase content of the respective organ. These results suggest that the cGMP kinase concentration of nonsmooth muscle tissues depends on the amount of organ-specific vascular smooth muscle and may be used as an index for the vascularization of these organs.

Animals↗
PubMed1989

[Rubella, measles, mumps--epidemiologic, occupational medicine significance and efficacy of adult vaccination].

A total of 437, 565 and 1.642 sera collected from personnel of Freiburg university hospital were tested for antibodies to measles, mumps and German measles. Susceptibility was found in about 10-30% of all cases. 107 persons were vaccinated. Seroconversions could be demonstrated in 80% (mumps) and 95.5% (German measles) of sera collected upon vaccination. Our results clearly demonstrate that mumps, measles, and German measles cannot be regarded as "childhood diseases" and hospital personnel is at increased infection risk.

Adult↗
PubMed1989

[The value of the tuberculin tine test].

The sensitivity of two different tuberculin multipuncture tests (PPD Merieux and Tubergen) was compared with Mendel Mantoux GT 10 in 213 persons. Merieux tests were false-negative in 15.3% of the cases, whereas congruence of the positive Mendel Mantoux and Tubergen reactions was 100%.

Adult↗
PubMed1988

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II.

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.

Animals↗
PubMed1988

Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea.

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.

Animals↗
PubMed1988

cAMP-dependent protein kinase rapidly phosphorylates serine- 687 of the skeletal muscle receptor for calcium channel blockers.

The cAMP-dependent phosphorylation of the 165-kDa subunit of the receptor for organic calcium channel blockers (CaCB-receptors) was studied. Tryptic peptide analysis showed that cAMP-dependent protein kinase phosphorylates rapidly a serine in one peptide. Up to three peptides containing phosphoserine and -threonine are phosphorylated in a 2-h incubation. The isolated 165-kDa subunit was digested with trypsin and the endoproteinase Lys-C and Glu-C. The rapidly phosphorylated peptide was isolated from each digest. The amino acid sequence was determined by Edman degradation and compared with the deduced amino acid sequence of the CaCB-receptor from rabbit skeletal muscle (Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 238, 313-318). Phosphoserine was determined as the phenylthiohydantoin-derivative of dithiothreitol-dehydroalanine. The phosphorylated serine was identified as Ser-687 which is localized between the transmembrane regions II and III. A second phosphopeptide was isolated into which phosphate was incorporated into Ser-1617 with a slow time course. This peptide is located in the COOH-terminal cytoplasmic domain of the 165-kDa subunit. It is anticipated that phosphorylation of serine 687 affects the opening probability of the calcium channel.

Animals↗
PubMed1988

The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein.

The cardiac receptor for calcium channel blockers was purified from bovine microsomal membranes which contained 235 +/- 33 fmol nimodipine-binding sites/mg protein (mean +/- SEM of nine preparations). To identify the receptor during the purification 20% of its binding sites were prelabeled with (+)[3H]PN200-110. The receptor was solubilized with 0.6% digitonin and was purified to a specific density of 157 pmol/mg using a combination of ion-exchange, wheat-germ-agglutinin-Sepharose chromatography and sucrose density gradient centrifugation. In the last sucrose gradient bound (+)[3H]PN200-110 comigrated with a 195-kDa protein. ( +/-)[3H]Azidopine and [3H]ludopamil, the photoaffinity ligands for the dihydropyridine and phenylalkylamine-binding site of the calcium channel, were incorporated specifically into the 195-kDa protein. These data indicate that the bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Its molecular mass suggests that the bovine cardiac receptor differs considerably from the rabbit skeletal muscle receptor protein for calcium channel blockers.

Affinity Labels↗
PubMed1988

Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase triggers acute morphological changes in thyroid epithelial cells.

In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.

Actins↗
PubMed1988

Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle.

Phospholamban of isolated sarcoplasmic reticulum of cardiac and smooth muscle is phosphorylated by cyclic GMP-dependent protein kinase (G-kinase). Concomitantly, the affinity of the Ca2+ pump for Ca2+ is increased. These effects are very similar to those seen with cyclic AMP-dependent protein kinase (A-kinase). The phosphate incorporation into phospholamban and the stimulatory effects of both kinases on the Ca2+ pump are not additive, suggesting that G-kinase phosphorylates the same serine residue as A-kinase. A possible physiological role for phosphorylation of phospholamban by G-kinase is discussed.

Animals↗
PubMed1988

8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase.

The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4 degrees C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 microM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 microM CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 microM and 2 microM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.

Binding Sites↗
PubMed1988

Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine-binding sites.

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.

Affinity Labels↗