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E Uribe

Publications and source records attributed to E Uribe.

12 recordsLinked to original sources
PubMed1999

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Arginase↗
PubMed1999

Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase.

Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.

Catalysis↗
PubMed1999

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.

Catalysis↗
PubMed1995

Interaction of arginase with metal ions: studies of the enzyme from human liver and comparison with other arginases.

As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.

Arginase↗
PubMed1995

[Phase II trial with navelbine + cisplatin + etoposide in the treatment of inoperable non-small cell lung carcinoma].

40 patients with advanced non-small cell lung cancer not previously treated were included in a study at phase II with vinorelbine 20 mg/Sq.m days 1 and 8, etoposide 60 mg/sq.m days 1-3 and cisplatin 75 mg/sq.m day 1 each 28 days for 6 cycles. There were 31 men and 9 women, being the average age of 54 years, with "performance Status" grade 0-2. All of them could be evaluated for toxicity and 31 for responsiveness. 10 patients were in stage IIIb and 21 at stage IV. 42% of objective responses were obtained and an overall survival of 9 months, which justifies further studies.

Adult↗
PubMed1989

[Gammagraphy with 111In-labelled leukocytes in an acute outbreak of inflammatory intestinal disease. Evaluation of the localization, extension and degree of activity].

We have studied 59 patients suspected of presenting an acute bout of inflammatory bowel disease: 23 had Crohn's disease and 36 had ulcerative colitis. All them underwent scintigraphic examination using 111In labelled leukocytes during the first 72 hours after their admittance and in a period no longer than 30 days, they also underwent barium enema and/or endoscopic study. Scintigraphic examination was more sensitive (100%), specific (100%) and accurate (100%) than endoscopy (72%, 100% and 78%) and barium enema (69%, 80% and 71%) in Crohn's disease. In ulcerative colitis, although endoscopy (100%) was slightly more sensitive than scintigraphy (100% and 94% against 78% and 93%). Reliability of barium enema was always lower to that of scintigraphy. In both conditions, scintigraphic examination detected a higher number of affected segments than barium enema did (40/16 in Crohn's disease and 65/31 in ulcerative colitis). Correlation between the index of scintigraphic activity and Harvey's index of clinical and biologic activity was highly significant (p less than 0.001) in both diseases. We conclude that scintigraphic examination using 111In labelled leukocytes is a reliable examination method for evaluation of localization, extension, and degree of activity of inflammatory bowel disease and that it must be a part of the examination protocol.

Acute Disease↗
PubMedDate not supplied

Manganese-dependent inhibition of human liver arginase by borate.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

Arginase↗