Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N-terminal 670 residues, which contain the active sites, and a unique 500-residue C-terminus highly enriched in proline, glycine, and alanine. The C-terminal region contains a second actin-binding site which allows myosins IA and IB to cross-link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin-binding site that activates ATPase. Myosin II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled-coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29-residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.
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PMID:Structure-function studies on Acanthamoeba myosins IA, IB, and II. 327 84

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.
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PMID:Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins. 331 20

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.
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PMID:Autophosphorylation of smooth-muscle caldesmon. 341 67

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.
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PMID:Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase. 352 65

The 20,000-dalton light chain of turkey gizzard myosin is phosphorylated at two sites. Dual phosphorylation is observed when both intact myosin and isolated light chains are used as substrates. Phosphorylation of the second site is not observed at higher ionic strength (e.g. 0.35 M KCl). The first phosphorylation site (serine 19) is phosphorylated preferentially to the second site. The latter is phosphorylated more slowly than the first site, and its phosphorylation requires relatively high concentrations of myosin light chain kinase. It is suggested that myosin light chain kinase catalyzes the phosphorylation of both sites on the light chain, and several reasons are cited that make it unlikely that a contaminant kinase is involved. The second phosphorylation site is a threonine residue. Based on the results of limited proteolysis of the light chain, it is concluded that the threonine residue is close to serine 19, and possible locations are threonines 9, 10, and 18. At all concentrations of MgCl2, phosphorylation of the second site markedly increases the actin-activated ATPase activity of myosin and accelerates the superprecipitation response of myosin plus actin.
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PMID:Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. 383 10

Phospholipids were found to be a constant component of rat glomerular basement-membrane preparations. The concentration fell during preparation of basement membrane by sonication of whole glomeruli, but then remained constant despite continued sonication. The proportions of the individual phospholipids were different from those of whole renal tissue or of isolated glomeruli. The basement-membrane preparations had no (Na(+)+K(+))-activated adenosine triphosphatase activity, an enzyme that is bound to plasma membranes. The concentration of lipid P was decreased on exposure in vivo or in vitro to antiserum against basement membrane; 7 days after injection of antiserum there was a change in the phospholipid composition, with a relative increase in phosphatidylcholine and a decrease in sphingomyelin content. The metabolic turnover rate of the lipid P remaining in the membrane was normal, as determined by (32)P incorporation. The loss of phospholipid was associated with decreases in the relative concentrations of hydroxyproline, hydroxylysine and glycine, and relative increases in proline, lysine, serine, threonine and valine. Administration of aminonucleoside and daunomycin produced proteinuria but did not cause a decrease in lipid P. Anticollagen and anti-lymphocyte sera that attached to the basement membrane but failed to produce proteinuria, also failed to affect the phospholipid content.
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PMID:Phospholipid of the rat glomerular basement membrane in experimental nephrosis. 426 92

A mutant (KF11) of Escherichia coli H+-translocating ATPase (F1-F0) has a single point mutation in the beta subunit of F1 that has lost 90% of its Mg2+-dependent ATPase activity (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703). The mutation was mapped at about the 500th nucleotide residue from the 5' end of the beta subunit gene by a genetic recombination test on the physical map of the cistron coding for the beta subunit. The mutant allele of KF11 (uncD11) was cloned on a hybrid plasmid (pKF11) via DNA isolated from a lambda uncD11 transducing phage. Restriction fragments of pKF11 containing the estimated mutation site were subjected to polyacrylamide gel electrophoresis under conditions where strands were separated into single strands. The two strands of a DNA segment, which was shown to carry an altered base, showed anomalous migration compared with those from the wild-type fragment. The results confirmed the result of mapping of the altered site by genetic tests. On the basis of these results, the nucleotide sequence of the mutated gene was determined, and a single base change of the 524th cytosine to thymine resulting in a phenylalanine for serine substitution at residue 174 of the beta subunit was found. This result, together with results on the altered properties of F1 from KF11 reported previously, indicates that residue 174 is essential for the Mg2+-dependent ATPase activity of F1 but not for the Ca2+-dependent ATPase activity.
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PMID:A phenylalanine for serine substitution in the beta subunit of Escherichia coli F1-ATPase affects dependence of its activity on divalent cations. 608 79

Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58-60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum.
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PMID:Physical and enzymatic properties of myosin from porcine brain. 611 56

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).
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PMID:Degradation of smooth-muscle myosin by trypsin-like serine proteinases. 612 14

Evidence now exists for the phosphorylation of all the major proteins of the myofibril with the exception of troponin C. Although uncertainty exists in most cases about the role of phosphorylation of the myofibrillar proteins, there is substantial evidence that phosphorylation of serine 20 of rabbit cardiac troponin I leads to a lowering of the sensitivity of the actomyosin ATPase to Ca2+. This process is of special importance in the physiological response of the heart to adrenalin. A well defined enzymic system involving a specific kinase and a phosphatase is present in most muscles for the phosphorylation and dephosphorylation of the P light chain (regulatory, L2 or DTNB light chain) of myosin. Myosin light-chain kinase is very active in fast skeletal muscles, and although it is unlikely that phosphorylation followed by dephosphorylation of the P light chain occurs fast enough to be synchronous with the contractile cycle, phosphorylation may have a modulatory role in this tissue. Both post-tetanic potentiation and the reduced actomyosin ATPase turnover rate observed in fast-twitch muscle as a consequence of sustained forceful contraction have been suggested by different investigators to be consequences of P light chain phosphorylation. Nevertheless, unequivocal evidence associating either of these effects with phosphorylation is not yet available. Kinase activity is also high in vertebrate smooth muscle and it has been suggested that phosphorylation of the P light chain is the process that activates the actomyosin ATPase in this tissue. Evidence from a number of studies indicates, however, that regulation of smooth muscle actomyosin ATPase may not be a simple phosphorylation-dephosphorylation process.
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PMID:Phosphorylation of the myofibrillar proteins and the regulation of contractile activity in muscle. 613 9


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