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K Bárány

Publications and source records attributed to K Bárány.

At least 19 recordsLinked to original sources
PubMed2001

Exchange of the actin-bound nucleotide in intact arterial smooth muscle.

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In (32)P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [gamma,beta-(32)P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the beta-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the gamma-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K(+), as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.

Actins↗
PubMed1995

Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

Animals↗
PubMed1993

Calponin phosphorylation does not accompany contraction of various smooth muscles.

Calponin phosphorylation was quantitated in 32P-labeled porcine arterial, uterine, tracheal, stomach and bladder smooth muscles. The negligible amount of 0.003-0.008 mol [32P]phosphate/mol calponin in resting muscles did not increase upon contraction induced by various agents, under conditions when myosin light-chain phosphorylation increased several-fold. The results indicate no involvement of calponin phosphorylation is smooth-muscle contraction.

Animals↗
PubMed1992

Involvement of calponin and caldesmon in sustained contraction of arterial smooth muscle.

The molecular mechanism of smooth muscle contraction was approached by a novel method, covalent 14C-labeling. Intra- and intermolecular protein interactions during contractile activity are reflected by changed reactivity of protein side chains; these can be detected by reagents which readily permeate through the muscle membrane without affecting the contractility and form covalent bonds with proteins in the muscle. The incorporation of 14CH2ICONH2 into proteins of 1-hour histamine contracted versus resting porcine carotid arterial muscles was determined. Out of fourteen 14C-labeled proteins analyzed, only two showed a change in reactivity during sustained contraction. The incorporation of 14CH2ICONH2 into calponin and caldesmon in contracted muscles was about 66% of that into these same proteins in resting muscles. A transformation of calponin and caldesmon molecules from an extended to a more compact conformation explains the decreased reactivity.

Animals↗
PubMed1992

Protein phosphorylation during the contraction-relaxation-contraction cycle of arterial smooth muscle.

Porcine carotid arterial muscles were labeled with 32P and then subjected to a resting-contraction-relaxation-contraction cycle. Four different agents were used for contraction: KCl, histamine, norepinephrine, and phorbol dibutyrate. To relax the contracted muscles, they were washed with physiological salt solution. Changes in the [32P]phosphate content of four different proteins--myosin light chain, a 28-kDa cytosolic protein, desmin, and caldesmon--were followed. In a short contraction-relaxation-contraction cycle lasting minutes, induced by K+, histamine, or norepinephrine, only the light chain underwent a phosphorylation-dephosphorylation-rephosphorylation without concomitant cyclic phosphorylation of the 28-kDa protein, desmin, or caldesmon. In a contraction-relaxation-contraction cycle of long duration, 60-min contractions with K+, histamine, or norepinephrine, cyclic phosphorylation of both the light chain and desmin was observed. With 60-min phorbol dibutyrate stimulation, in the long contraction-relaxation-contraction cycle, the phosphorylations of the light chain, desmin, and caldesmon were cycling. It is concluded that under physiological conditions, light-chain phosphorylation initiates both short and sustained arterial contraction. Desmin phosphorylation is likely to be involved in force maintenance during sustained contraction.

Animals↗
PubMed1992

Protein phosphorylation in arterial muscle contracted by high concentration of phorbol dibutyrate in the presence and absence of Ca2+.

Porcine carotid arterial muscles were 32P-labeled then contracted with 8 microM phorbol dibutyrate (PDBu) in normal physiological salt solution (PSS) and in Ca(2+)-free PSS containing 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetate. Significant incorporation of [32P]phosphate into the 20-kDa myosin light chain, a 28-kDa protein, desmin and caldesmon was measured with no apparent difference between normal and Ca(2+)-depleted muscles. Ca-determination showed that the Ca(2+)-depleted muscle contained 15% of the total Ca of the normal muscle. However, determination of the actin-bound Ca revealed that all the Ca in the Ca(2+)-depleted muscle could be accounted for by its actin-bound Ca. Accordingly, protein phosphorylation during the slow PDBu-induced contraction may proceed in the virtual absence of free Ca2+. Phosphopeptide mapping of the myosin light chain isolated from muscles contracted with PDBu either in the presence or absence of Ca2+ showed that two-thirds of the incorporated [32P]phosphate was attributable to myosin light chain kinase catalyzed phosphorylation and one-third was due to phosphorylation by protein kinase C. PDBu increased the phosphorylation of the 28-kDa protein, desmin and caldesmon two- to threefold, as compared with that in muscles contracted by KCl depolarization or by the receptor mediated agonists norepinephrine and histamine. Muscles contracted by high concentration of PDBu in the presence or absence of Ca2+ could be fully relaxed and recontracted.

Animals↗
PubMed1991

Exchange of 20-kDa myosin light chain-bound phosphate during sustained contraction of arterial smooth muscle.

K(+)-contracted porcine carotid arterial muscles containing phosphorylated 20-kDa myosin light chains (LC) were exposed to carrier-free [32P]orthophosphate in K(+)-stimulating solution during sustained contraction. The covalently bound LC phosphate was completely replaced by [32P]phosphate, indicating that myosin light chain phosphatase and kinase have ready access to the bound phosphate during the sustained contraction. On average, 0.38 mol [32P]phosphate was incorporated per mole LC during the sustained K+ contraction. This value was about half of the maximal value for [32P]phosphate incorporation into LC, 0.74 mol/mol, in muscles contracted with K+ for 1 min. Assuming that sustained contraction involves the maximal number of cross-bridges attached to actin, the data suggest that half of the attached cross-bridges contain phosphorylated LC.

Animals↗
PubMed1991

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate.

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.

Animals↗
PubMed1991

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle.

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 265, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 32P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [32P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.

Animals↗
PubMed1990

Stretch activates myosin light chain kinase in arterial smooth muscle.

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

Animals↗
PubMed1990

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery.

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.

Animals↗
PubMed1989

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle.

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.

Animals↗
PubMed1989

Effects of ATP reduction on the pattern of force development and myosin light chain phosphorylation in intact arterial smooth muscle.

The effect of reduction of ATP content on phosphorylation of the 20 kDa light chain of myosin (MLC) and force development in intact carotid arterial smooth muscle was investigated. With reduction of ATP to 23% of control by treatment with 2-deoxyglucose there was reduction in basal, in peak and 30 min MLC phosphorylation during contraction (P less than 0.001). The rate of force development was reduced, but maximal force was the same as control. By treatment with 50 microM iodoacetate, the resting ATP content was unchanged but fell to 22% after 30 min contraction. Basal MLC phosphorylation was the same as control, but peak (P less than 0.001) and 30 min phosphorylation were lower (P less than 0.005), even though the rate and magnitude of force development were greater. The results indicate that neither rate nor magnitude of force development correlate with MLC phosphorylation. Basal and initial MLC phosphorylation may play a cooperative role in contractile function.

Adenosine Triphosphate↗
PubMed1988

Phosphorylation of the 20,000-Da myosin light chain isoforms of arterial smooth muscle by myosin light chain kinase and protein kinase C.

Phosphorylation of myosin light chain (LC) isoforms in arterial actomyosin can be induced by endogenous kinases upon addition of Mg2+ and ATP. The extent of phosphorylation in the presence of 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 0.2 mM Ca2+, or 0.2 mM Ca2+ plus calmodulin is 1.6, 2.1, and 2.3 mol phosphate/mol LC, respectively. Two-dimensional gel electrophoresis of actomyosin shows that the LC isoforms may be mono-, di-, and triphosphorylated. Tryptic phosphopeptide mapping of LC indicates that the radioactive phosphate is distributed to six peptides referred to as A through F. Phosphoamino acid analyses and the phosphopeptide maps of isolated LC, phosphorylated by either purified myosin light chain kinase or protein kinase C, reveal that myosin light chain kinase phosphorylates a serine residue in peptides A and B, and threonine plus serine residues in peptides C and D. Peptides E and F are phosphorylated by protein kinase C in serine and threonine residues, respectively. In actomyosin, with EGTA, phosphorylation of peptides E and F proceeds while phosphorylation of peptides A, B, C, and D is inhibited. Ca2+ and calmodulin enhance the phosphorylation of peptides A, B, C, and D, while phosphorylation of peptides E and F is decreased. In isolated LC, myosin light chain kinase preferentially phosphorylates the peptides A and B over C and D. Phosphorylation of peptides E and F in LC by protein kinase C promotes additional phosphorylation of peptides C and D by myosin light chain kinase, whereas phosphorylation of peptides A and B is diminished. The present data suggest that the phosphorylation of distinct sites in arterial myosin light chain by myosin light chain kinase and protein kinase C is interrelated.

Adenosine Triphosphate↗
PubMed1988

Effect of okadaic acid on phosphorylation-dephosphorylation of myosin light chain in aortic smooth muscle homogenate.

Myosin light chain phosphorylation in aortic smooth muscle homogenate reached a maximal level of 0.75 mol phosphate/mol light chain, and then declined. Addition of okadaic acid led to a sustained phosphorylation level of 1.7 mol/mol. In the absence of okadaic acid, phosphorylation was predominantly due to myosin light chain kinase, whereas in the presence of okadaic acid both myosin light chain kinase and protein kinase C were involved in phosphorylation. Okadaic acid inhibited dephosphorylation of the distinct sites in LC phosphorylated by either myosin light chain kinase or protein kinase C, suggesting that it exerts its effect through inhibition of myosin light chain phosphatases present in aortic homogenate.

Animals↗
PubMed1987

Evidence for isoforms of the phosphorylatable myosin light chain in rat uterus.

The phosphorylatable myosin light chain of rat uterus was resolved into four spots by two-dimensional gel electrophoresis. Antibody against the 20 kDa light chain from smooth muscle myosin recognized these four spots. Purified light chain exhibited four spots on a diagonal upon isoelectrofocusing in both first and second dimensions, proving that these spots are not due to artifactual charge modification. Accordingly, the four spots contain light chain isoforms derived from myosin.

Animals↗
PubMed1987

Comparison of myosin light chain phosphorylation in uterine and arterial smooth muscles.

1. The phosphorylatable light chain from uterine and arterial smooth muscles appear as four spots on two-dimensional gel electrophoretograms due to the existence of isoforms which may be non-, mono- or diphosphorylated. 2. The phosphorylation sites are serine and threonine residues; the phosphoserine to phosphothreonine ratio is smaller, and the extent of diphosphorylation is larger in uterus than in artery. 3. Different phosphorylation values found at identical tension levels and identical phosphorylation values found at different tension levels narrow the role of light chain phosphorylation to the activation of smooth muscle contraction.

Animals↗