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M D Pato

Publications and source records attributed to M D Pato.

At least 37 records · Page 2Linked to original sources
PubMed1987

Characterization of the smooth muscle phosphatases and study of their function.

The activities of some proteins involved in the process of contraction-relaxation in smooth muscle cells are regulated by reversible phosphorylation. Phosphorylation of myosin by MLCK has been shown to be a pre-requisite for muscle contraction. MLCK, itself, is a substrate for cAMP-dependent protein kinase. Relaxation is favored in the event that MLCK is phosphorylated by cAMP-dependent protein kinase because this modification inhibits the activity of MLCK. In our attempt to understand the mechanism and regulation of contractile activity in smooth muscle cells, we purified and characterized the enzymes which catalyze the dephosphorylation of myosin and MLCK. We have purified 3 smooth muscle phosphatases termed SMP-I, II and IV to apparent homogeneity and partially purified SMP-III from turkey gizzards. Characterization of these enzymes revealed that they are distinct. They have different physical, enzymatic and immunological properties. As isolated, all 4 enzymes dephosphorylate myosin light chains rapidly but only SMP-III and IV are active toward myosin or heavy meromyosin. However, SMP-I could be activated toward myosin when its catalytic subunit is dissociated from the regulatory subunits and when the 55,000-Da regulatory subunit is digested or released from the holoenzyme. Recently we have purified to apparent homogeneity 2 protein phosphatases from rabbit uterine muscle. Partial characterization of these enzymes revealed their close similarity to the avian smooth muscle phosphatases. Analysis of the properties of the smooth muscle phosphatases led us to speculate on their function in vivo. SMP-III and IV are most likely to dephosphorylate myosin to cause relaxation because they exhibit the highest activity toward intact myosin. SMP-I may play a role in this process if there is a physiological mechanism which dissociates the catalytic subunit from the 55,000-Da regulatory subunit or from both regulatory subunits. A more obvious role for SMP-I is to dephosphorylate MLCK following phosphorylation by cAMP-dependent protein kinase to restore the high activity of MLCK. SMP-II does not dephosphorylate myosin and has low activity toward MLCK. It is active toward glycogen synthase suggesting a role in glycogen metabolism for the production ATP required to supply the energy for contraction. We are currently undertaking experiments to verify these proposals.

Animals↗
PubMed1986

Limited proteolytic digestion and dissociation of smooth muscle phosphatase-I modifies its substrate specificity. Preparation and properties of different forms of smooth muscle phosphatase-I.

Smooth muscle phosphatase-I (SMP-I), a protein phosphatase purified from turkey gizzard smooth muscle, is composed of 2 regulatory subunits (Mr = 60,000 and 55,000) and a catalytic subunit (Mr = 38,000). Two other forms of this enzyme have been prepared and characterized. The free catalytic subunit, termed SMP-Ic, was prepared by ethanol treatment of SMP-I, and a form devoid of the 55,000-Da subunit, termed SMP-I2, was prepared by limited tryptic digestion. Exposure of SMP-I to proteases like trypsin and chymotrypsin results in a rapid degradation of the 55,000-Da polypeptide. Degradation of the catalytic subunit is observed only upon prolonged digestion. The 60,000-Da polypeptide appears to be resistant to the action of trypsin and chymotrypsin. SMP-I dephosphorylates myosin light chains but is not active toward intact myosin or heavy meromyosin. However, when the catalytic subunit is dissociated from both regulatory subunits or from the 55,000-Da polypeptide, the enzyme becomes active toward myosin suggesting that the 55,000-Da polypeptide inhibits the activity of the catalytic subunit toward myosin. In addition to alteration of the substrate specificity, the regulatory subunits also modulate the effect of divalent cations, like Mn2+, on the activity of the enzyme.

Animals↗
PubMed1985

Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards.

A phosphoprotein phosphatase that dephosphorylates smooth muscle myosin has been purified to apparent homogeneity from turkey gizzards. Smooth muscle phosphatase (SMP) IV has a molecular weight of 150,000 as determined by gel filtration on a Sephadex G-200 column and is composed of two subunits (Mr = 58,000 and 40,000). Although it is active toward a number of proteins, its activities toward the contractile proteins, intact myosin, heavy meromyosin, and isolated myosin light chains are higher than its activities toward phosphorylase alpha, histone IIA, and phosphorylase kinase. SMP-IV preferentially dephosphorylates the beta-subunit of phosphorylase kinase. The properties of the enzyme have been studied using heavy meromyosin, a soluble chymotryptic fragment of myosin, and isolated myosin light chains as substrates. SMP-IV has high affinity for both substrates and is optimally active at neutral pH. Divalent cations, Ca2+ and Mg2+, activate the dephosphorylation of heavy meromyosin but inhibit the activity toward myosin light chains. Low concentrations of ATP (1-5 mM) activate SMP-IV but concentrations higher than 5 mM are inhibitory. Inhibition of 50% of the activity of the enzyme by NaF and PPi requires concentrations higher than 10 mM. Rabbit skeletal muscle heat stable inhibitor-2 has no effect on the activity of SMP-IV toward heavy meromyosin, myosin light chains, and phosphorylase alpha.

Animals↗
PubMed1985

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers.

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.

Animals↗
PubMed1984

The binding of smooth muscle myosin light chain kinase and phosphatases to actin and myosin.

Contractile activity in smooth muscle cells is regulated by phosphorylation-dephosphorylation of the 20,000-Da light chain of myosin. In an attempt to better understand the localization in muscle of the enzymes which catalyze the phosphorylation-dephosphorylation process, we measured the binding constants of turkey gizzard smooth muscle myosin light chain (MLC) kinase and smooth muscle phosphatases (SMP) to myosin and actin under identical conditions by a sedimentation method. We have observed that MLC kinase binds strongly to both actin and myosin. When tropomyosin is complexed to actin, the affinity of MLC kinase to actin increases 2-3-fold. The presence of calcium-calmodulin weakens the binding of MLC kinase to actin, actin-tropomyosin, and myosin by about 3-fold. Increasing the ionic strength of the binding assay also decreases the binding of MLC kinase to myosin and actin-tropomyosin. MLC kinase is observed to bind to rod subfragment, a fragment of myosin which does not contain the phosphorylatable light chain suggesting that the kinase also binds to domains of the myosin other than the 20,000-Da light chain. Of the phosphatases tested, only SMP-III and -IV bind strongly to unphosphorylated myosin. When the myosin is thiophosphorylated , the binding constants of SMP-III and -IV increase dramatically. SMP-I and -II do not bind to unphosphorylated and thiophosphorylated myosin. However, the free catalytic subunit of SMP-I binds weakly to thiophosphorylated myosin. None of the phosphatases binds to actin. Our study suggests that in muscle, the myosin phosphatase is localized in the thick filament while the MLC kinase may be associated with the thick filaments, thin filaments, or even both.

Actins↗
PubMed1983

Purification and characterization of a multisubunit phosphatase from turkey gizzard smooth muscle. The effect of calmodulin binding to myosin light chain kinase on dephosphorylation.

A phosphatase that is active in dephosphorylating the isolated 20,000-Da light chain of myosin, as well as the enzyme myosin light chain kinase, has been purified to apparent homogeneity from turkey gizzards. The enzyme has a molecular weight of 165,000 by sedimentation-equilibrium centrifugation under nondenaturing conditions and is composed of three subunits (Mr = 60,000, 55,000, and 38,000) in a 1:1:1 molar ratio. The properties of the holoenzyme, as well as the purified catalytic subunit (Mr = 38,000) were compared using myosin light chains, intact myosin, and myosin light chain kinase as substrates. Although the holoenzyme is active in dephosphorylating the isolated myosin light chains and the enzyme myosin light chain kinase, the holoenzyme does not dephosphorylate myosin. On the other hand, the catalytic subunit of the holoenzyme dephosphorylates all three substrates. When myosin light chain kinase, which has been phosphorylated at two sites is used as substrate, both sites are rapidly dephosphorylated by the phosphatase in the absence of bound calmodulin. If calmodulin is bound to the diphosphorylated kinase, only one site is dephosphorylated. Interestingly, the single site dephosphorylated when calmodulin is bound to myosin light chain kinase is the site that is not phosphorylated when the calmodulin-myosin kinase complex is phosphorylated by cAMP-dependent protein kinase.

Animals↗
PubMed1983

Characterization of a Mg2+-dependent phosphatase from turkey gizzard smooth muscle.

A Mg2+-dependent phosphatase has been purified to apparent homogeneity from turkey gizzard smooth muscle. The enzyme has a Mr = 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 44,500 as determined by sedimentation equilibrium centrifugation under nondenaturing conditions. Using polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate all of the phosphatase activity was found to migrate as a single band, subsequently shown to have an Mr = 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is inactive in the absence of Mg2+ and maximum activity is reached at a free concentration of 12 mM Mg2+. Mn2+ can replace Mg2+, but the activity is only about one-fifth of that found with 12 mM Mg2+. NaF and the nucleotides ATP, ADP, and AMP inhibit phosphatase activity. This inhibition appears to be independent of their ability to bind Mg2+. The phosphatase purified from turkey smooth muscle appears to be identical with that purified from canine heart (Binstock, J. F., and Li, H. C. (1979) Biochem. Biophys. Res. Commun. 87, 1226-1234) and rat liver (Hiraga, A., Kikuchi, K., Tamura, S., and Tsuiki, S. (1981) Eur. J. Biochem. 119, 503-510).

Adenine Nucleotides↗
PubMed1983

The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein phosphatase-2A2.

Two homogeneous protein phosphatases, termed 'smooth muscle phosphatase-I' and 'smooth muscle phosphatase-II', isolated from turkey gizzard as enzymes active against the 20-kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purified as an enzyme active against protein synthesis initiation factor eIF-2, were classified using the criteria defined by Ingebritsen and Cohen [Eur. J. Biochem. (1983) 132, 255-261]. All three enzymes were type-2 protein phosphatases based on their specificity for the alpha-subunit of phosphorylase kinase and insensitivity to inhibitor-1 and inhibitor-2. The substrate specificities of smooth muscle phosphatase-I and the eIF-2 phosphatase were similar to the catalytic subunit of protein phosphatase-2A. Smooth muscle phosphatase-I could be designated as protein phosphatase-2A1 and eIF-2 phosphatase as protein phosphatase-2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase-II allowed its assignment as protein phosphatase-2C.

Animals↗
PubMed1982

Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP.

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.

Adenosine Triphosphatases↗
PubMed1982

Regulation of contractile proteins by reversible phosphorylation of myosin and myosin kinase.

In vitro experiments support the ideal that the actin-activated MgATPase activity of smooth muscle myosin and myosin from nonmuscle cells is regulated by the phosphorylation of the 20,000 dalton light chain of myosin. Experiments with intact smooth muscles support this mechanism but also raise the possibility that tension may be maintained in the presence of partial dephosphorylation (12). The possibility that smooth muscle contraction may also be modulated by additional regulatory systems (13,29) is to be expected based on experience with other types of muscle. The enzyme myosin light chain kinase catalyzes the phosphorylation of the 20,000 dalton light chain of myosin. This enzyme requires Ca2+-calmodulin for activity. The activity of myosin kinases that have been isolated from avian smooth muscle cells (8) or human platelets (16) can be decreased by phosphorylation. This phosphorylation is catalyzed by cAMP-dependent protein kinase and decreases myosin kinase activity by interfering with the binding of Ca2+-calmodulin. A number of different phosphatases have been purified from smooth muscle (22). These phosphatases play an important role in determining the state of phosphorylation of myosin and myosin kinase. Two areas of particular interest at present are the regulation of phosphatase activity and the physiological significance of myosin kinase phosphorylation.

Animals↗
PubMed1981

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin.

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.

Adenosine Triphosphatases↗
PubMed1981

Fragments of rabbit striated muscle alpha-tropomyosin. I. Preparation and characterization.

Fragments of muscle alpha-tropomyosin have been prepared by limited and prolonged tryptic digestion, by limited chymotryptic digestion, and by chemical cleavage procedures at the methionine and single cysteine residues. Procedures are described for their isolation in a homogeneous form and for their identification by amino acid, NH2-terminal, and COOH-terminal analyses. The initial site of tryptic cleavage was at Arg-133, close to Asp-137, the only such acidic residue to occur in a "core" a or d position of the coiled-coil structure. Chymotrypsin cleaved initially at Leu-169. Both sites of initial cleavage are close to minima in the averaged alpha-helical periodicity previously reported. Trypsin-resistant fragments were isolated corresponding to residues 13-125 and 183-284, indicating that the NH2-terminal region (1-13) and central region (126-183) are more susceptible to proteolytic degradation. Comparison of the thermal stabilities of the fragments showed that all fragments from the COOH-terminal half of the molecule were less stable than those from the NH2-terminal half. These stability differences could be related to differences in the averaged alpha-helical potentials of the fragments estimated from their amino acid compositions. It is suggested that these differences in stability properties reflect considerable variation in the nature and affinity of the actin-binding sites as well as an imposition on the COOH-terminal half of the molecule of structural requirements for the binding of troponin.

Amino Acid Sequence↗
PubMed1981

Fragments of rabbit striated muscle alpha-tropomyosin. II. Binding to troponin-T.

The interactions of a variety of large fragments of rabbit skeletal muscle alpha-tropomyosin, prepared as previously described, with troponin-T and a soluble tropomyosin-binding fragment of troponin-T (CB1) have been investigated by affinity chromatography and gel filtration. No specific interactions between NH2-terminal fragments encompassing residues 1-189 with troponin, troponin-T, or CB1 immobilized on a Sephadex 4B column could be demonstrated. Similarly, there was no interaction between these fragments and CB1 on a gel filtration column operated in 0.1 M KCl, 10 mM imidazole pH 7.0 buffer. On the other hand, all fragments encompassing residues 190-284 showed interaction with troponin-T on the affinity column and with CB1 by gel filtration. When mixtures of two fragments, one of which had the intact NH2-terminal sequence of the original tropomyosin structure and the other the intact COOH-terminal sequence, were applied to the gel filtration column, there was no indication of interaction between them. However, when CB1 was included in the mixture, a ternary complex of the three components was demonstrable. Fragments in which 10 or 12 residues at the NH2-terminal end of the alpha-tropomyosin sequence were absent showed no evidence of forming a ternary complex with CB1 and the COOH-terminal fragments. We conclude that the binding of the troponin-T fragment, CB1, to the COOH-terminal third of the alpha-tropomyosin molecule enhances head-to-tail aggregation of tropomyosin molecules either indirectly by the transmission of conformational changes to the head-to-tail overlap region or more directly by binding close to or at this region.

Animals↗
PubMed1980

Regulation of myosin light chain kinase by reversible phosphorylation and calcium-calmodulin.

1) Myosin light chain kinases from smooth muscle and platelets can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. 2) Phosphorylation of both kinases, in the absence of calmodulin, markedly decreases kinase activity. 3) The decrease in smooth muscle myosin kinase activity is due to a decreased affinity of the phosphorylated kinase for calmodulin. 4) Dephosphorylation of the smooth muscle kinase by a phosphatase isolated from smooth muscle restores the affinity of the kinase for calmodulin.

Animals↗