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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.
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PMID:Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella. 13 96

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.
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PMID:Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1). 13 45

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.
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PMID:Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles. 13 46

When sarcoplasmic reticulum vesicles are exposed to trypsin for 1 min the adenosine triphosphatase (Mr = 102,000) is cleaved to fragments of Mr = 45,000 and 55,000. The purified ATPase, containing both fragments, transports Ca2+ when incorporated into vesicles containing excess phospholipid. The two fragments can only be dissociated in solutions containing 1% sodium dodecyl sulfate (SDS). Ca2+ transport activity is restored in SDS-dissociated preparations in a series of steps involving dilution with 5 volumes of 5% phospholipids in 0.75% sodium cholate, incubation in ice for 30 min, and passage through an anion exchange column. Vesicles formed in this procedure regain high Ca2+ transport activity if they are incubated in SDS solution at 24 degrees for less than 20 min. However, the extent of renaturation diminishes if the vesicles are incubated for longer periods and little acitivity is recovered in vesicles incubated longer than 60 min at 24 degrees.
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PMID:Restoration of calcium transport in the trypsin-treated (Ca+ + Mg2+)-dependent adenosine triphosphatase of sarcoplasmic reticulum exposed th sodium dodecyl sulfate. 13 48

1. Isolated F1 (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 muM. 2. About one-third of the F1 and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles). 3. After further treatment with alkali of urea-treated particles, binding of F1 requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc2 are added. The concentration of F1-binding sites in the presence of both OSCP and Fc2 is about the same as that in TU particles. 4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F1, unless Fc2 is also present. Fc2 induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive. 5. It is tentatively concluded that OSCP is the binding site for F1 and Fc2 is the binding site for OSCP.
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PMID:Proteins required for the binding of mitrochondrial ATPase to the mitochondrial inner membrane. 13 85

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1.
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PMID:A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae. 13 3

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.
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PMID:Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin. 13 79

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.
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PMID:Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system. 13 62

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked. 2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase. 3. The molecular weight of the ATPase inhibitor is about 10,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition. 4. The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP. 5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation. 6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.
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PMID:Isolation and characterization of an inhibitory subunit of the Mg2+--Ca2+-ATPase of Escherichia coli. 13 64

The adenosine 5'-triphosphate (ATP)-linked transhydrogenase reaction, present in the particulate fractions of Escherichia coli, was previously shown to be inhibited in these fractions when the bacteria were treated with colicins K or El. The purpose of this study was to characterized the ATP-linked transhydrogenase reaction and the colicin-caused inhibition of the reaction in purified cytoplasmic membranes. Particulate fractions from bacteria treated or untreated with colicins were separated on sucrose gradients into cell wall membrane and cytoplasmic membrane fractions. The ATP-linked transhydrogenase reaction was found to be exclusively associated with the cytoplasmic membrane fractions. The reaction was inhibited by carbonylcyanide m-chlorophenlhdrazone, dinitrophenol, N,N'-dicyclohexylcarbodiimide, and trypsin. Although the cytoplasmic membrane fractions were purified from the majoriy of the cell wall membrane and its bound colicins, they showed the inhibitory effects of colicins K and El on the ATP-linked transhydrogenase reaction. The inhibition of ATP-linked transhydrogenase reaction induced by the colicin could not be reversed by subjection the isolated membranes to a variety of physical and chemical treatments. Cytoplasmic membranes depleted of energy-transducing adenosine triphosphatase ATPase) complex (coupling factor) lost the ATP-linked transhydrogenase activity. The ATPase complexes isolated from membranes of bacteria treated or untreated with colicins El or K reconstituted high levels of ATP-linded transhydrogenase activity to depleted membranes of untreated bacteria. The same ATPase complexes reconstituted low levels of activity to depleted membranes of the treated bacteria.
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PMID:Adenosine 5'-triphosphate-linked transhydrogenase in cytoplasmic membranes of colicin-treated and untreated Escherichia coli. 13 1


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