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G Bailin

Publications and source records attributed to G Bailin.

At least 19 recordsLinked to original sources
PubMed1996

Reaction of rabbit skeletal myosin with D-glucose 6-phosphate.

Incubation of rabbit skeletal myosin with 1 to 3 mM D-glucose 6-phosphate over a period of several hours resulted in the inhibition of the K(+)- and actin activated-ATPase activities. Substrate ATP (0.5-3 mM final concentration) protected the myosin against the loss of ATPase activity as induced by glucose 6-phosphate. This was also found for ADP. When the myosin was incubated with 3 mM [3H] labeled glucose 6-phosphate for 28 h. up to one mole of glucose 6-phosphate was incorporated per 4.7 x 10(5) g of myosin. A significant reduction in the labeling occurred in the presence of ATP. The labeling was limited to the heavy chain region as judged by gel electrophoresis which resolved the heavy and light chain components of myosin. The non-enzymatic glycation of myosin by glucose 6-phosphate is probably the primary cause for the observed loss of the ATPase activity of myosin. This effect may also occur physiologically modifying the activity of muscle contractile proteins particularly during prolonged hyperglycemia.

Actins↗
PubMed1994

Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca2+ + Mg2+)- ATPase protein of sarcoplasmic reticulum at low temperature.

Modification of the (Ca2+ + Mg2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, at 4 degrees C for 5 min caused a 63% loss of the Ca(2+)-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10(5) g of protein. At 25 degrees C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca(2+)-ATPase activity was inhibited to the same extent. MgATP protected the ATPase activity at 4 degrees C and 25 degrees C but there was little change in the extent of labeling at 4 degrees C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4 degrees C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca(2+)-ATPase activity and only a small decrease in the labeling occurred. At 25 degrees C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4 degrees C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A2 subfragment to the A1 subfragment in the absence of deoxycholate and from the A1 subfragment to the A2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Fluorescence changes of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to thiol groups of gizzard myosin.

The fluorescence intensity of gizzard myosin that incorporated about 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) at 0.6 M KC1 was quenched at low concentrations of MgATP or in the presence of F-actin. The observed quenching was reduced at high MgATP concentrations. The half maximal change in quenched fluorescence was shifted from 7.5 microM to 25 microM MgATP for NBD-myosin at 0.15 M KC1. The fluorescent label NBD-C1 can be used to detect changes in the conformation of gizzard myosin.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Fluorescence properties of the Ca2+,Mg2(+)-ATPase protein of sarcoplasmic reticulum labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

The fluorescence intensity of the Ca2+,Mg2(+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum that incorporated about 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was enhanced at high MgATP concentrations with or without 50 microM calcium. The observed enhancement indicates that the fluorophore, NBD-Cl, can detect conformational changes in the ATPase protein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.

4-Chloro-7-nitrobenzofurazan↗
PubMed1986

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin.

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.

Animals↗
PubMed1985

Reaction of phenylglyoxal with chicken gizzard myosin.

Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.

Adenosine Triphosphatases↗
PubMed1984

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase.

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.

Adenosine Triphosphatases↗
PubMed1984

Dinitrophenylation of phosphorylated gizzard myosin. Labelling of the subfragment 1 region.

Chymotrypsin and papain digestion of 3H-labelled dinitrophenylated chicken gizzard myosin, pretreated with the myosin light chain catalyzed phosphorylating system, released a subfragment 1 devoid of its light chains but containing all of the label associated with the thiols of the heavy chain region. This was also the case when myosin was incubated in the presence of the phosphorylating system but without ATP. Dinitrophenylation of a reconstituted actomyosin made from phosphorylated myosin occurred mainly on -SH groups of the heavy chains, in contrast to the predominant modification of the light chains of myosin from a control actomyosin that was treated similarly. Conformational changes in myosin may govern, in part, actin-myosin interaction through the phosphorylation of the light chain of Mr 20.000.

Actomyosin↗
PubMed1983

Peptide fragments of the heavy-chain region of phosphorylated and dinitrophenylated gizzard myosin.

Sulfopropyl (SP) Sephadex chromatography of trypsin-pepsin digests of dinitrophenylated gizzard myosin, pretreated with and without the myosin light-chain kinase calcium-calmodulin phosphorylating system, yielded similar elution patterns and nine peptide fractions were found. From a comparison with trypsin-pepsin digests of the heavy chains of dinitrophenylated myosin, pretreated with and without the phosphorylating system, it was established that peptide I, a major peptide fraction, was part of the 17-kDa dinitrophenylated light chain. Phosphorylation of myosin did not change the dinitrophenyl group content of peptide I but it did result in a significant increase in the dinitrophenylation of other peptides. The peptides contained only S-dinitrophenyl cysteine. Peptide III, previously considered to be part of the light-chain region (Bailin, G. and Lopez, F. (1982) J. Biol. Chem. 257, 264-270), was shown to originate in the heavy chains of myosin based on a comparison of the elution patterns of the digests of modified myosin and its heavy chains. Several neutral and basic peptides (peptides III to IX) originated in the heavy-chain region and they were different from those from the heavy chains of rabbit skeletal myosin. Phosphorylation of the 20-kDa light chain shifted the dinitrophenylation of the sulfhydryl groups from the 17-kDa light chain to the heavy chains of myosin, predominantly. These thiol groups do not resemble the fast-reacting -SH groups of rabbit skeletal myosin. The light chains are involved, in part, in making sites available on myosin that are necessary for actin-myosin interaction.

Adenosine Triphosphate↗
PubMed1983

Effect of phosphorylation and dinitrophenylation on chicken gizzard myosin.

Treatment of phosphorylated chicken gizzard myosin which had incorporated 1.5 mol of phosphate per 4.7 x 10(5) g of protein with 1-fluoro-2,4-dinitrobenzene resulted in the modification of the heavy and light chains when 5.8 mol of the reagent were bound to myosin. Concurrently, the K+-ATPase activity was inhibited and the modified myosin possessed actin activated-ATPase activity. Thiolysis of nearly 2 mol of the dinitrophenyl group mainly from the heavy chains (and some light chains) of the modified myosin with 2-mercaptoethanol restored the K+-ATPase activity. Digestion of phosphorylated gizzard myosin with chymotrypsin or papain occurred to a lesser extent than a control myosin. Chymotryptic fragments of phosphorylated and dinitrophenylated myosin were formed at a faster rate than those of dinitrophenylated myosin alone suggesting that phosphorylation of the light chain of Mr 20,000 altered the susceptibility of the heavy chains of myosin to proteolysis. Phosphorylation of dinitrophenylated gizzard myosin which had incorporated 5.5 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein was the same as that of a control myosin; this was also the case for the thiolyzed dinitrophenylated myosin. In the absence of calcium, phosphorylation of control and dinitrophenylated myosins decreased by 73% suggesting that the phosphorylation reaction was calcium dependent. Phosphorylation and dinitrophenylation induced conformational changes in the light chains of gizzard myosin that may be involved in maintaining the structure of the heavy chain region.

Adenosine Triphosphatases↗
PubMed1981

Dinitrophenylation of chicken gizzard myosin: reactivity of the 17 000-dalton light chain.

Chicken gizzard myosin rapidly incorporated 3 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein with little change in the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity. During an interval when 2 additional mol of the reagent were bound the K+-ATPase activity in the presence of EDTA was inhibited and the Ca2+-ATPase activity was altered to a lesser extent. Cysteine residues were modified in the dinitrophenylated gizzard myosin. The dinitrophenyl group was located mainly in the active proteolytic fragment, subfragment 1. Dinitrophenylation of the heavy and light chains was observed but major changes in the ATPase activity occurred when the 17 000-dalton light chain and some heavy chains were modified as judged by dissociation experiments in sodium dodecyl sulfate. Thiolysis of the dinitrophenylated gizzard myosin with 2-mercaptoethanol restored the ATPase activity and approx. 2 mol of the dinitrophenyl group were removed. The restoration of the enzymic activity, however, occurred when 1 mol of the label was thiolytically cleaved from cysteine residues of the 17 000-dalton light chain. Substrate Mg-ATP(2-) or MgADP did not protect the ATPase activity of modified gizzard myosin. In the presence of nucleotide there was an increase in the incorporation of the reagent, and a change in its distribution into the light and heavy chains. Calcium had no effect on the dinitrophenylation of this myosin. these results indicate that the reagent, 1-fluoro-2,4-dinitrobenzene, could detect chemical differences in smooth muscle myosin when compared to the reactivity of other myosins. Thiol groups of the 17 000-light chain (and some heavy chains) are probably located peripheral to the active site region of gizzard myosin and they are involved in maintaining the enzymic activity of this protein.

Adenosine Triphosphatases↗
PubMed1981

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene.

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.

Adenosine Triphosphate↗
PubMed1980

Crosslinking of sarcoplasmic reticulum ATPase protein with 1,5-difluoro 2,4-dinitrobenzene.

The reacton of 1,5-difluoro-2,4-dinitrobenzene with the ATPase protein of rabbit skeletal sarcoplasmic reticulum caused a marked loss of the Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity during an interval when 2 mol of the crosslinking reagent were incorporated/10(5) g of protein. The modified ATPase protein formed non-serial high molecular weight aggregates or oligomers during short (1--5 min) or long exposure (60 min) to the reagent at 25 degrees C or 4 degrees C. The same pattern was found when sarvoplasmic reticulum was treated similarly; only the ATPase protein formed oligomers (homopolymers). In all cases the ATPase protein monomer remained the predominant species present. During the appearance of the high molecular weight oligomers the Ca2+-ATPase activity was unaffected but Ca2+ uptake was inhibited. Major changes in the ATPase activity occurred when the monomeric ATPase protein was modified. Disubstituted dinitrophenylene derivatives of cysteine and tyrosine were found in modified ATPase protein and only a small amount of monosubstituted dinitrophenyl groups were identified. Thiolysis of the modified ATPase protein with 2-mercaptoethanol removed approx. 35% of the incorporated groups, but there was no restoration of the Ca2+-ATPase activity. Substrate MgATP2- protected the Ca2+-ATPase activity of the ATPase protein and sarcoplasmic reticulum but Ca2+ had no effect on the modificaton. Different conformational states of the ATPase protein could be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the bifunctional reagent dinitrophenylation of the ATPOase protein with that of the monofunctional reagent 1-fluoro-2,4-dinitrobenzene (Bailin, G. (1980) Biochim. Biophys. Acta 623, 213-224). Intramolecular crosslinking of the ATPase protein predominated and oligomers which formed during the reaction were not essential for the maintenance of the ATPase activity.

Adenosine Triphosphatases↗
PubMed1980

Dinitrophenylation of rabbit skeletal sarcoplasmic reticulum ATPase protein.

The ATPase (ATP phosphohydrolase (EC 3.6.1.3)) protein of rabbit skeletal sarcoplasmic reticulum rapidly incorporated three mol of 1-fluoro-2,4-dinitrobenzene per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-dependent ATPase activity was inhibited. The dinitrophenyl group was located mainly in the ATPase protein and a small amount of the label was found in the proteolipid component of the ATPase preparation as judged by dissociation experiments in sodium dodecyl sulfate. Cysteine and tyrosine residues were dinitrophenylated in the modified ATPase protein. Thiolysis of the dinitrophenylated ATPase protein with 2-mercaptoethanol under various conditions did not restore the Ca2+-dependent ATPase activity. Solubilization of the ATPase protein with sodium deoxycholate increased the reactivity of the reagent and the Ca2+-dependent ATPase activity was inhibited to a greater extent. Dinitrophenylation of the ATPase protein was Ca2+-dependent; in the presence of high Ca2+ the incorporation increased by 50% and a large decrease in the Ca2+-ATPase activity was noted. The half-maximal changes for the incorporation of the reagent and the inhibition of the Ca2+-ATPase activity occurred at 3--4 microgram Ca2+-concentration, consistent with the binding of Ca2+ to high affinity sites on the ATPase protein. These results indicate that the ATPase protein as a Ca2+-free and a Ca2+-bound conformation. The reagent, 1-fluoro-2,4-dinitrobenzene reacts differentially and thus characterizes these two conformations.

Adenosine Triphosphatases↗