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

---

Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.
...
PMID:Uncoupling the red cell sodium pump by proteolysis. 300 82

Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.
...
PMID:Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane. 301 34

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.
...
PMID:Accessibility of tryptophan residues in Na,K-ATPase. 303 Oct 29

Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2-1 micron in diameter containing a high density of oriented Na,K-ATPase molecules are bound to a planar lipid bilayer acting as a capacitive electrode. In the aqueous phase adjacent to the bound membrane sheets, ATP is released within milliseconds from an inactive, photolabile precursor ("caged" ATP) by an intense flash of light. After the ATP-concentration jump, transient current and voltage signals can be recorded in the external circuit corresponding to a translocation of positive charge across the pump protein from the cytoplasmic to the extracellular side. These electrical signals which can be suppressed by inhibitors of the Na,K-ATPase require the presence of Na+ but not of K+ in the aqueous medium. The intrinsic pump current Ip(t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system. Ip(t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasi-stationary current. The time constant of the rising phase of Ip(t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic origin of the current transient can be obtained by double-flash experiments and by chymotrypsin modification of the protein. These and other experiments indicate that the observed charge-translocation is associated with early events in the normal transport cycle. After activation by ATP, the pump goes through the first steps of the cycle and then enters a long-lived state from which return to the initial state is slow.
...
PMID:Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP. 304 Oct 2

Nonstationary pump currents which have been observed in K+-free Na+ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na+ precedes translocation of K+. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a "dielectric coefficient" describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E1-P/P-E2 conformational transition of the protein, followed by a release of Na+ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E1-P/P-E2 transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.
...
PMID:Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents. 304 Oct 3

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.
...
PMID:Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct. 316 Jun 95

Previous studies have shown that the erythrocyte membrane Ca2+ pump is exposed primarily to the cytoplasm: proteases, substrates and polyclonal antibodies all interact with the enzyme from the cytoplasmic side. In this study, the pump's accessibility from outside the cell was investigated with monoclonal antibodies. When cultures of hybridoma cells producing antibodies against the Ca2+ pump were screened for binding of the antibodies to intact red cells, only 7% of the cultures gave a positive reaction (a total of eight cultures). The small number of positives confirms the relative inaccessibility of the Ca2+ pump from outside the red cell. From the eight positive cultures we isolated one stable clone which produced an antibody (1B10) that reacted both with purified Ca2+ pump and with the outside of intact red cells. Immunoprecipitation experiments and binding assays with inside-out vesicles showed that 1B10 reacted only against the erythrocyte Ca2+ pump from the extracellular face of the red cell. 1B10 had no observable effect on the Ca2+ efflux from resealed red cells. Digestion of intact red cells with glycosidases, trypsin or papain had minimal effect on the binding of the antibody to intact red cells. However, digestion with pronase, subtilisin or alpha-chymotrypsin nearly eliminated the binding, indicating that 1B10 was directed against a protein determinant of the ATPase which is exposed on the outside of the red cell.
...
PMID:Topology of the erythrocyte Ca2+ pump. A monoclonal antibody against the almost inaccessible extracellular face. 320 39

The B-protein of phage Mu, which is required for high frequency intermolecular transposition in vivo, shows ATPase activity in vitro, binds nonspecifically to DNA, and stimulates intermolecular strand transfer. To elucidate the structural bases for B-protein function, it was subjected to limited proteolysis with two different proteases, trypsin and chymotrypsin. The resulting fragments were mapped by amino acid sequencing. These data show that the B-protein is organized in two domains: an amino-terminal domain of 25 kDa and a carboxyl-terminal domain of 8-kDa. A fragment analogous to the amino-terminal domain, produced by deleting the 3' end of a cloned B gene, proved to be insoluble and had to be renatured after elution from a sodium dodecyl sulfate gel. The renatured protein retains ATP-binding activity and to a lesser extent the DNA-binding activity of the MuB protein, but is unable to hydrolyze ATP or function in transposition. We also show in this study that efficient DNA-strand transfer by the B-protein occurs even in the absence of a detectable ATPase activity or in the presence of adenosine 5'-O-(thio)triphosphate (ATP gamma S).
...
PMID:Structure-function relationships in the transposition protein B of bacteriophage Mu. 329 29

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.
...
PMID:Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins. 331 20

The hypothesis that the ADP-sensitive form of phosphorylated Na+, K+-ATPase contains occluded sodium ions has been tested by a procedure which involves (i) modifying the enzyme with alpha-chymotrypsin or N-ethylmaleimide (NEM) so that the ADP-sensitive form is more stable than it is in the native enzyme, (ii) phosphorylating the modified enzyme with ATP in the presence of labelled sodium ions, and (iii) forcing the phosphorylated enzyme rapidly through a cation-exchange column and measuring the labelled sodium in the effluent. The results show that ADP-sensitive phosphoenzyme prepared from alpha-chymotrypsin- or NEM-modified Na+, K+-ATPase is able to carry labelled sodium ions through a cation-exchange resin. This behaviour was not seen with native Na+, K+-ATPase or when phosphorylation was prevented by the omission of magnesium ions or by the substitution of adenylyl(beta, gamma-methylene)diphosphonate (AMP-PCP) for ATP. The occluded sodium ions were rapidly released when the phosphoenzyme was dephosphorylated by ADP. When alpha-chymotrypsin-modified enzyme was phosphorylated by ATP with 1 mM-sodium in the medium, close to three sodium ions were occluded per phospho group. The stoicheiometry at much lower sodium concentrations could not be determined satisfactorily. A consideration of the rate constants of the reactions thought to be involved in the occlusion of sodium and in the release of sodium from the occluded state shows that, so far as they are known, these constants are compatible with the hypothesis that the occluded-sodium form of the phosphoenzyme plays a central role in sodium transport through the pump.
...
PMID:The occlusion of sodium ions within the mammalian sodium-potassium pump: its role in sodium transport. 608 5


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---