Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When initial velocities are measured with yeast hexokinase at pH 7, 17 degrees, the inert coordination complex chromium-ATP is competitive vs. MgATP and noncompetitive with glucose, with a dissociation constant of 4-6 muM in either the presence or absence of glucose. These patterns confirm a random kinetic mechanism for this enzyme. With CrATP present, however, the reaction slows down over the first several minutes to a much slower rate, suggesting tighter binding of CrATP with time. When CrATP, MgATP, and D-lyxose are preincubated with the enzyme for 10 min and the reaction started by addition of excess glucose, the dissociation constand of CrATP in now 0.13 muM and the reaction is linear with time. When glucose, CrATP, and enzyme are incubated together and then placed on a Sephadex column, 1 mol each of CrATP and glucose per active center is tightly bound to the enzyme, thus providing a simple and precise method of determining the concentration of active sites. This tight complex, after denaturation with acid, releases 25% free glucose and 75% of a chromium complex containing both ADP and sugar-6-P. CrADP-glucose-6-P is also slowly released from the enzyme during incubation, so that CrATP is actually a very slow substrate. Binding of CrATP with the formation of CrADP-sugar-6-P complexes is also induced by mannose, fructose, glucosamine, 2,5-anhydro-D-glucitol, 2,5-anhydro-D-mannose, and 2,5-anhydro-D-mannitol, while glucose-6-P, 6-deoxyglucose, and lyxose also induce tight binding of CrATP. With excess enzyme, only 25% of CrATP is bound, and the rest does not inhibit the hexokinase reaction. Since bidentate Cr(NH3)4ATP and monodentate CrADP also display inhibition which is tighter with time, but since bidentate CrADP is a poor inhibitor, the actural substrates in the hexokinase reaction appear to be beta, gamma-bidentate MgATP and beta-monodentate MgADP. Tighter inhibition by Cr-8-BrATP than by CrATP suggests that ATP ASSUMES THE SYN CONFORMATION ON THE ENZYME. The substrate inhibition by MgATP induced by the presence of lyxose is shown to be competitive vs. glucose and partial, and, together with other data available, to suggest a kinetic mechanism that is random, but where (1) the rate constant for release of glucose from E-glucose is equal to Vmax, and that for release of glucose from central complexes is less than Vmas; (2) the majority of the reaction flux when both substrates are present at Km levels goes through the path with glucose adding before MgATP, but where at physiological levels the flux through the two paths is more equal. Contd.
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PMID:Use of chromium-adenosine triphosphate and lyxose to elucidate the kinetic mechanism and coordination state of the nucleotide substrate for yeast hexokinase. 108 14

1. We studied the non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the L-arginine (L-ARG)/nitric oxide (NO) pathway in this response. 2. Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin-1 (ET-1), relaxed frequency-dependently in response to electrical stimulation. Maximum relaxation was obtained at 6-8 Hz, and amounted to 56 +/- 2%, 77 +/- 3% and 62 +/- 6% of the agonist-induced tension in preparations contracted by NA, carbachol, or ET-1, respectively. Exposure to NG-nitro-L-arginine (L-NOARG; 10(-7)-10(-5) M) concentration-dependently reduced the relaxant response in preparations contracted by NA. L-NOARG (10(-6) M) reduced the maximal response to 51 +/- 8% of control. L-NOARG (10(-5) M) abolished all relaxation, and unmasked a contractile component; D-NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET-1, L-NOARG (10(-5) M) markedly reduced relaxations evoked by electrical stimulation. 3. In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with L-ARG (10(-3) M), and the inhibitory effect of L-NOARG (10(-6) M) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies less than 6 Hz, but a small inhibition was observed at higher frequencies. 4. Administration of NO (present in acidified solution of NaNO2) induced concentration-dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET-1.L-NOARG (10-5 M) and L-ARG (10-3M) had no effect on these relaxations. However, methylene blue (10-S M) significantly shifted the concentration-response curve for NO to the right. NANC-relaxation and NO-induced relaxation of trigonal preparations were both inhibited by oxyhaemoglobin (10-5 M) and pyrogallol (10-4 M).5. In urethral preparations precontracted by NA, electrical stimulation caused frequency-dependent relaxations. A maximum relaxation of 73 +/- 4% was obtained at 10 Hz. Also in the urethra, NANCrelaxation was blocked by L-NOARG (10-5 M), and a contractile response generally appeared.6. Detrusor strips treated with alpha-beta methylene ATP (10-i M) and atropine (10-6 M), and then contracted by ET-1, showed relaxations (19 +/- 3% of the induced tension) in response to electrical field stimulation (2-20 Hz) only when the tension was high. No response at all, or small contractions, were found in response to electrical stimulation in K+ (35 mM)-contracted detrusor strips. Detrusor preparations contracted by carbachol were concentration-dependently relaxed by exogenously administered NO, SIN-1 (NO-donor), and isoprenaline, whereas vasoactive intestinal polypeptide had minor effects. NO and SIN-1 induced maximal relaxations of 63 +/- 3% and 70 +/- 4%, respectively, of the tension induced by carbachol. Isoprenaline produced an almost complete relaxation (96 +/- 4%).7. The results suggest that NANC-nerve mediated relaxation, involving the L-ARG/NO pathway, can be demonstrated consistently in the pig trigonal and urethral, but not in detrusor smooth muscle. The importance of this pathway for lower urinary tract physiology and pathophysiology remains to be established.
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PMID:Nitric oxide and relaxation of pig lower urinary tract. 139 68

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.
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PMID:Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy. 167 Jul 78

The expression of protein-tyrosine kinases (PTKs; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) was studied in normal human lung and various tumors by PCR followed by molecular cloning and sequence analysis. Six known PTKs (YES, FGR, LYN, HCK, PDGFB-R, and CSF1-R), as well as two additional members of this enzyme family, were detected in lung. One of the newly discovered sequences appears to represent a group of cytosolic PTKs. The cDNA sequence of the second unknown PTK revealed that it is a fourth member of the fibroblast growth factor receptor family. It was therefore called TKF (tyrosine kinase related to fibroblast growth factor receptor). Among a wide variety of cells and tissues tested, including human lymphocytes and macrophages, TKF was only found expressed in lung. Apart from normal lung, TKF expression could be demonstrated in some tumors of lung origin, but also in malignancies not derived from lung tissues. As fibroblast growth factors are generally involved in a variety of functions such as mitogenesis, angiogenesis, and wound healing, the specific expression of a receptor-related gene in lung only may point to yet another special function of this group of proteins.
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PMID:Two additional protein-tyrosine kinases expressed in human lung: fourth member of the fibroblast growth factor receptor family and an intracellular protein-tyrosine kinase. 172 May 39

The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.
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PMID:Baculovirus expression of functional P210 BCR-ABL oncogene product. 249 63

Forty male STD-Wistar rats, weighing about 210 g on the average, were divided into two dietary groups. These were further subdivided into the following eight groups: 1) control rats fed the normal diet (N rats: group #1), N rats treated with 0.1 mg Cd (group #2), 0.5 mg Cd (group #3), and 1.0 mg Cd (group #4); 2) Mg-deficient rats (D rats: group #5), D rats treated with 0.1 mg Cd (group #6), 0.5 mg Cd (group #7), and 1.0 mg Cd (group #8). Before Cd treatment the rats were given the normal diet or the Mg-deficient diet for 14 consecutive days day -14). Subcutaneous injection of 0.1 ml of cadmium chloride (CdCl2) in the backs of the animals given a normal diet or a Mg-deficient diet at the three doses of 0.1, 0.5, and 1.0 mg/kg body was performed twice a day (12-h intervals) (time-zero) for 7 consecutive days and then these animals were maintained without Cd treatment for an additional period of 20 days (+28 days). Body weight gain in Mg-deficient rats (D rats) was significantly decreased. The effects of Cd treatment in the rats fed the normal diet (N rats) were also significant. Mg deficiency enhanced the decreased body weight gain in D rats treated with Cd on day 24 though no enhancement of the decreased food consumption in those rats was observed. Mg deficiency lowered the blood pressure in rats and this response was more pronounced in D rats treated with Cd. The increased urinary Na excretion and the decreased water retention were not observed in the D rats; this response was not pronounced in the D rats treated with Cd. These results suggest that an enhancement of the decreased blood pressure in Cd-treated rats by the Mg deficiency is not responsible for the decreased water retention. Ca concentration in the heart and aorta of D rats was within the same range as that of N rats. Mg deficiency increased Ca concentration in the heart and aorta of the D rats treated with 0.5 and 1.0 mg Cd though Cd itself did not affect the Ca and ATP concentrations, Ca and Mg balance (Ca/Mg), and heart weight in the heart of N rats. These results suggest that Mg deficiency may increase the overload of Ca in the heart and aorta of D rats treated with Cd, which may in turn lead to enhancement of the Cd-induced cardiotoxic effects.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effect of Mg deficiency on blood pressure in rats treated with cadmium. 344 86

We used phosphorus-31 nuclear magnetic resonance to test the ability of a perfluorocarbon blood substitute that has been shown in previous studies to improve oxygen delivery to hypothermic myocardium to maintain aerobic high-energy phosphate metabolism during total global ischemia. Twenty-three isolated perfused rabbit hearts were subjected to 180 min of hypothermic (23 degrees C) global ischemia followed by 45 min of normothermic reperfusion. Hearts received multiple doses of a cardioplegic solution that contained either oxygenated perfluorocarbon (Fluosol O2), nonoxygenated perfluorocarbon (Fluosol N2), or standard crystalloid hyperkalemic cardioplegic solution (STD-KCl) at 30 min intervals. Recovery of isovolumic left ventricular developed pressure (LVDP) was used to assess preservation of contractile function. Recovery of LVDP was 84 +/- 19% of preischemic control values with Fluosol O2, 68 +/- 16% with Fluosol N2, and 67 +/- 17% with STD-KCl (p = .058 vs Fluosol N2 and p = .056 vs STD-KCl). During 3 hr of ischemia intracellular pH (pHi) fell to 6.68 +/- 0.20 with STD-KCl and to 6.71 +/- 0.14 with Fluosol N2 but remained above 7.00 throughout the ischemic period with Fluosol O2 (p less than .0001 vs Fluosol N2 or STD-KCl). Myocardial ATP content was better preserved at 107 +/- 14% of control values with Fluosol O2 compared to 60 +/- 18% of control with Fluosol N2 and 75 +/- 21% of control with STD-KCl (p less than .001 vs Fluosol N2, p = .002 vs STD-KCl). Phosphocreatine (PCr) was also better preserved with Fluosol O2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Maintenance of aerobic metabolism during global ischemia with perfluorocarbon cardioplegia improves myocardial preservation. 669 19

In rat liver epithelial cell lines (WB or GN4), angiotensin II (Ang II) stimulates cytosolic tyrosine kinase activity, in part, through a calcium-dependent mechanism. In other cell types, selected hormones that activate Gi- or Gq-coupled receptors stimulate the soluble tyrosine kinase, p125FAK. Immunoprecipitation of p125FAK from Ang II-activated GN4 cells demonstrated a doubling of p125FAK kinase activity. However, an additional Ang II-activated tyrosine kinase (or kinases) representing the majority of the total activity was detected when the remaining cell lysate, immunodepleted of p125FAK, was reimmunoprecipitated with an anti-phosphotyrosine antibody. Cytochalasin D pretreatment blocks G-protein receptor-dependent tyrosine phosphorylation in Swiss 3T3 cells. While cytochalasin D decreased the Tyr(P) content of 65-75-kDa substrates in Ang II-treated GN4 cells, it did not diminish tyrosine phosphorylation of 115-130-kDa substrates, again suggesting activation of at least two tyrosine kinase pathways in GN4 cells. To search for additional Ang II-activated enzymes, we used molecular techniques to identify 20 tyrosine kinase sequences in these cell lines. None was the major cytosolic enzyme activated by Ang II. Specifically, JAK2, which had been shown by others to be stimulated by Ang II in smooth muscle cells, was not activated by Ang II in GN4 cells. Finally, we purified Tyr(P)-containing tyrosine kinases from Ang II-treated cells, using anti-Tyr(P) and ATP affinity resins; 80% of the tyrosine kinase activity migrated as a single 115-120-kDa tyrosine-phosphorylated protein immunologically distinct from p125FAK. In summary, Ang II activates at least two separate tyrosine kinases in rat liver epithelial cells; p125FAK and a presumably novel, cytosolic 115-120-kDa protein referred to as the calcium-dependent tyrosine kinase.
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PMID:Angiotensin II activates at least two tyrosine kinases in rat liver epithelial cells. Separation of the major calcium-regulated tyrosine kinase from p125FAK. 749 50

JAK family tyrosine kinases have recently been implicated in intracellular signal transduction by transmembrane cytokine receptors of the interferon (IFN) and hematopoietin receptor families. Using the prolactin (PRL)-dependent rat pre-T cell line Nb2, a PRL receptor-associated, candidate tyrosine kinase of 120-130 kDa was recently characterized (1). In the present work this protein is identified as JAK2, based upon reciprocal anti-JAK2 and anti-phosphotyrosine immunoprecipitation and immunoblotting. JAK2 underwent rapid and transient tyrosine phosphorylation in response to receptor activation, reaching peak levels within 5 min of exposure to 100 nM PRL at 37 degrees C. In vitro tyrosine kinase assays using either [gamma-32P]ATP and autoradiography or unlabeled ATP combined with anti-phosphotyrosine immunoblotting, demonstrated that the activity of JAK2 was stimulated by PRL. Phosphoamino acid analysis of JAK2 after in vitro tyrosine kinase assay revealed that the majority of phosphate was incorporated into tyrosine residues. Furthermore, JAK2 was associated with PRL receptors to a comparable extent before and after PRL binding, as demonstrated by anti-receptor immunoprecipitation and subsequent anti-JAK2 immunoblotting. We propose that binding of ligand to the PRL receptor activates preassociated JAK2, and that this enzyme generates the initial signal in the intracellular communication cascade.
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PMID:Activation of receptor-associated tyrosine kinase JAK2 by prolactin. 750 35

One of the earliest cellular responses to prolactin (PRL) binding in Nb2 cells, a rat pre-T lymphoma cell line, is an increase in tyrosine phosphorylation of cellular proteins. In this work, immunologic techniques have been used to demonstrate that in Nb2 cells and in mouse mammary gland explants, JAK2, a non-receptor tyrosine kinase, is activated following stimulation with PRL. PRL stimulated tyrosine phosphorylation of JAK2 at times as early as 30 sec and concentrations of PRL as low as 0.5 ng/ml (2.5 pM) in Nb2 cells and 100 ng/ml (5 nM) in mammary gland explants. When JAK2 was immunoprecipitated from solubilized Nb2 cells or mammary gland explants and incubated with [gamma-32P]ATP, 32P was incorporated into a protein migrating with an apparent molecular weight appropriate for JAK2 only when cells had been incubated with PRL, indicating that JAK2 tyrosine kinase activity is exquisitely sensitive to PRL. In Nb2 cells, JAK2 was found to associate with PRL receptor irrespective of whether or not the cells had been incubated with PRL. These results provide strong evidence that JAK2 is constitutively associated with the PRL receptor and that it is activated and tyrosine phosphorylated upon PRL binding to the PRL receptor. These results are consistent with JAK2 serving as an early, perhaps initial, signaling molecule for PRL.
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PMID:Activation of JAK2 tyrosine kinase by prolactin receptors in Nb2 cells and mouse mammary gland explants. 751 93


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