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

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
...
PMID:Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities. 12 30

Sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals in the rabbit skeletal muscle sarcolemma the presence of four major protein bands corresponding to molecular weight 216,000, 110,000, 44,000, 15,000, and smaller amounts of 148,000, 78,000, 68,000, 37,000, 27,000 proteins. (Na+-K+)-ATPase isolated from rabbit skeletal muscle contains 102,000, 44,000, and 32,000 protein bands. Sarcolemma is resistant to the solubilizing effect of Triton X-100, Lubrol, and deoxycholate. Sarcolemmal proteins, especially sodium-potassium-ATPase, are sensitive to digestion with trypsin.
...
PMID:Characterization of sarcolemma from rabbit skeletal muscle. 13 Jun 57

Myosin purified from the abdominal flexor muscle of the lobster, Homarus americanus, has a number average length of 1559 +/- 218 A, a rod like tail 1335 A long and a globular head 225 X 45 A as determined from electron microscopic observations on platinum shadowed preparations. The mass of the molecule was determined to be ca. 486,000 daltons from high speed equilibrium centrifugation studies at neutral and alkaline pH, and by SDS-acrylamide gel electrophoresis. Both sedimentation equilibrium centrifuge studies at alkaline pH and SDS-acrylamide gel electrophoresis experiments, indicate that the molecule contains a heavy chain core (two polypeptide chains weighing ca. 210,000 daltons each) and ca. four light chains of two weight classes (ca. 16,000 and 20,000 daltons). The amino acid composition of the myosin was determined. The specific activities of the Mg2+ -activated, K+/EDTA-activated, and Ca2+ -activated ATPases of the myosin were determined. Kinetic analysis of the digestion of lobster myosin with trypsin suggests that lobster myosin contains three classes of lysine and arginine residues; slowly split (k = 2.07 +/- 0.31 X 10(-2) moles/min2), rapidly split (k = 11.0 +/- 1.83 X 10(-2) moles/min2) and trypsin insensitive. There are 187 +/- 22 slowly split residues, 280 +/- 35 rapidly split residues, and 144 +/- 41 trypsin insensitive bonds per molecule. Comparison of these molecular parameters with those for the vertebrate skeletal muscle myosin indicates that the two myosins are similar in terms of mass, shape and overall polypeptide chain composition but may be considerably different in terms of local polypeptide chain conformation or composition.
...
PMID:Comparative studies on the structure and aggregative properties of the myosin molecule. I. The structure of the lobster myosin molecule. 13 6

Dipolid human fibroblast-rich tissues contain a macromolecule with a molecular weight between 30,000 and 50,000 daltons which will inhibit the proliferation of fibroblasts in the G1 phase of the cell cycle (i.e., inhibit both 3H-thymidine uptake as well as the normal increase in cell number). The inhibitor is destroyed by trypsin but not by ribonuclease or deoxyribonuclease, and it is thermolabile. It has an acid IEP. It is not cytotoxic, and its inhibitory activity appears to be completely reversible. This fibroblast endogenous inhibitor does not interfere with the proliferation of DNA synthesis by human lymphocytes, bronchial carcinoma cells, or HeLa cells. The activity does not appear to be species specific. Therefore, we suggest that it is quite possible that the control of fibroblast proliferation resides in a fibroblast chalone. Diploid human fibroblasts, in contrast to chicken or mouse fibroblasts or heteroploid fibroblasts in general, stringently require serum for their proliferation. All of this mitogenic activity of calf serum can be concentrated in a molecular weight range around 100,000 daltons by ultrafiltration. All of the mitogenic activity within this molecular weight class can be concentrated at a pH of 5.2 via isoelectric focusing, and all of the activity at this isoelectric point can be concentrated in one peak on preparative polyacrylamide gel electrophoresis. This latter material is homogeneous at three different pH's in analytical gel electrophoresis as well as in SDS electrophoresis. This purified serum mitogen for diploid human fibroblasts in vitro also works in vivo and represents as much as 0.5% of calf serum protein, albeit there is much less of this protein in adult cow or horse. It is composed of two equal subunits weighing about 60,000 daltons each and contains about 2 moles of sialic acid, one S-S bond, and 6 moles of hexose per subunit. There is a reciprocal relationship between the biological activity of fibroblast inhibitor and serum mitogen, but there is no apparent direct interaction between these two proteins. Addition of pure serum mitogen to diploid human fibroblasts in vitro results in the release of commensurable chalone activity into the medium and a reciprocal loss of mitogen from the medium. Therefore, we propose that serum contains a single macromolecule which competes with endogenous chalone on the surface of diploid human fibroblasts and that this functions as an anti-chalone for the fibroblast.
...
PMID:Circulating factors controlling cell proliferation. 13 64

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.
...
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

30-S dynein ATPase from Tetrahymena cilia was digested with trypsin (dynein: trypsin = 20:1, by weight) at 25 degrees C for 20 min, resulting in the release of a 12-S fragment possessing ATPase activity. The 12-S ATPase fraction obtained by sucrose gradient centrifugation contained several polypeptide chains as indicated by SDS gel electrophoresis. The largest chain was smaller than the subunit of 30-S dynein and almost the same size as 14-S dynein. On the other hand, when 14-S dynein was digested in a similar manner, its sedimentation value changed from 14 to 12 S, but the peak of ATPase activity was retained at 14 S, suggesting differences in amino acid sequences between the 30 and 14-S dyneins. When the time course of tryptic digestion of 30-S dynein was investigated in a trypsin:dynein ratio of 1:200, discrete fragmentation took place, producing an intermediate fragment of 24 S and the 12-S fragment. The 24-S fragment recombined with outer fibers to some extent, while the 12-S fragment lacked this ability. However, the 12-S fragment was somewhat stimulated to recombine with outer fibers in the presence of other components involved in the trypsin digest. The enzymatic characteristics of the 12-S fraction were different from those of 30-S dynein, especially the activity dependence on pH showing a typical bell-shaped curve.
...
PMID:Tryptic fragmentation of 30-S dynein from Tetrahymena cilia. 14 Jul 5

A partial purification of epidermal G2 chalone from crude epidermal cell extracts from hairless mice is described. The procedure involved the sequential use of ammonium sulphate precipitation, affinity chromatography and gel filtration. Mitosis-inhibiting activity at each stage in the purification was tested in an in vitro assay system employing human epidermoid carcinoma cells in exponential growth phase and Colcemid (Ciba) for arresting mitoses. This assay system is much more rapid and convenient than conventional in vivo systems. By the procedure described, a 10,000-fold material purification of the active component has been obtained. This corresponds to a 3,000-fold purification measured by protein content, and a 300-fold increase in mitosis-inhibiting activity per unit we;ght. The active component is acidic, contains sugar residues and has gel chromatographic properties characteristic of a substance with molecular weight of approximately 20,000. On SDS polyacrylamide gel electrophoresis, however, three weak bands were found. The active component is resistant to trypsin and protease and stable between pH 6.0 and 8.5. It is easily inactivated at pH values below 6.0. At the present stage of purification, several components other than the active one still remain in our material and further purification steps must be eventually employed.
...
PMID:Partial purification of epidermal G2 chalone. 14 51

A method for primary culture of ovine myometrial cells is described. After dissection, myometrium of ewe uteri was digested in trypsin and collagenase. The cells were preplated for 1 h at 37 degrees C. The non-attached cells were grown in appropriate medium supplemented with 2% fetal calf serum. They had a doubling time of 3 days, reached confluency at 10 days and did not exhibit contact inhibition. Cultures were maintained up to 22 days. Characterization of the cells was achieved by electron microscopy, analysis of myosin in cell extracts and assessment of hormone sensitivity. The cells were found to contain myofilaments, characteristic of smooth muscle. A high content of myosin (6--13%) was demonstrated on SDS-polyacrylamide gel electrophoresis: this was confirmed by ATPase activity assay. Cells responded to estradiol stimulation by increased protein synthesis, and bound [3H]estradiol in a specific and saturable way. These results suggest that myometrial cells grown in primary culture should provide a useful model for studying the hormonal control of contractile protein synthesis.
...
PMID:Myometrial cells in primary culture: characterization and hormonal profile. 15 21

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.
...
PMID:Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. 16 36

Intracytoplasmic A particles were analyzed by immunodiffusion and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) before and after enzymatic cleavage with trypsin. A common antigen in A particles was detected by antisera prepared against purified intracytoplasmic A particles, purified mouse mammary tumor virus (MMTV), and a purified MMTV core polypeptide (p28). Despite this correlation, no SDS--polyacrylamide band migrating at p28 was observed in purified intracytoplasmic A particles. However, after incubation with trypsin, A particles subjected to SDS--PAGE produced only two polypeptide bands. They were observed at p28 and p15-10. Ouchterlony analysis of the trypsin-cleaved A particles revealed no alteration in the antigenicity of the particles. These results suggested that some structural components of intracytoplasmic A particles are polypeptide precursors of MMTV core proteins.
...
PMID:Mouse mammary tumor virus polypeptide precursors in intracytoplasmic A particles. 16 80


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

---