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L D Graham

Publications and source records attributed to L D Graham.

27 records · Page 2Linked to original sources
PubMed1993

Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities.

In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.

Amino Acid Sequence↗
PubMed1993

Safety, efficacy, cost, and morbidity of laparoscopic versus open cholecystectomy: a prospective analysis of 228 consecutive patients.

Laparoscopic cholecystectomy has become the procedure of choice in most hospitals for the resolution of surgically treatable gallbladder disease. Few reports address the results of laparoscopic cholecystectomy in comparison to open cholecystectomy during the same time interval within the same institution. One hundred ninety-six laparoscopic cholecystectomies were performed from April 1990 through February 1991. Initial patient selection was restricted to elective procedures for chronic cholecystitis with expanded indications as experience was gained. Of the 196 cases, 11 required conversion to open cholecystectomy, leaving 185 laparoscopic cholecystectomies for comparison. During the same period, 82 open cholecystectomies were performed. Thirty-nine of these were complicated cases and would not have been considered for laparoscopic cholecystectomy early in the study, leaving 43 routine open cholecystectomies for comparative purposes. In the laparoscopic group, 1.1 per cent of the patients had major operative complications as opposed to the open group, which had none. There were no common bile duct injuries in either group. To provide a true cost-benefit analysis, a group of patients was identified that would qualify for elective, same-day admission for either an open or laparoscopic procedure. Laparoscopic cholecystectomy (LC) was performed on 70 patients, and open cholecystectomy (OC) was performed on 26 patients. A comparison of data from these groups showed no significant difference in age or sex. Hospitalization costs averaged $5,390 for the LC group versus $5,392 for the OC group. Postoperative hospital stay averaged 1.3 days for the LC group versus 3.7 days for the OC group (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholangiography↗
PubMed1992

Morbidity associated with long-term methotrexate therapy in juvenile rheumatoid arthritis.

To evaluate the adverse effects associated with long-term methotrexate (MTX) therapy in children with juvenile rheumatoid arthritis, we conducted a retrospective review of 62 patients with polyarticular juvenile rheumatoid arthritis, treated from 84 to 296 weeks with MTX weekly. Pulmonary function testing was performed before MTX therapy on 46 patients older than 6 years of age; 26 patients had serial pulmonary function testing, and no abnormalities were detected. In all 62 patients, liver function (alanine aminotransferase and aspartate aminotransferase activity) was monitored every 3 months. Transient liver function abnormalities developed in nine patients during treatment. Twelve patients underwent percutaneous liver biopsies after receiving 815 to 2980 mg of MTX; none had fibrosis or cirrhosis. Macrocytic anemia developed in one child receiving simultaneous long-term trimethoprim-sulfamethoxazole therapy and resolved after the trimethoprim-sulfamethoxazole was discontinued. No stomatitis or rashes were observed. Six patients were able to discontinue MTX therapy when their disease remitted; 56 continue MTX therapy. No child permanently discontinued MTX therapy because of an adverse effect. These data suggest that MTX may be better tolerated in children with juvenile rheumatoid arthritis than in adults with rheumatoid arthritis.

Adolescent↗
PubMed1991

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases.

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

Amino Acid Sequence↗
PubMed1990

Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase [pyruvate dehydrogenase (lipoamide), E1p, EC 1.2.4.1)] component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The dissociation constant (Ks) was estimated to be not less than 0.3 mM, exceeding the Km value (33 microM) for reductive acetylation of the domains by an order of magnitude. Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to E1p. The difference between Ks and Km suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.

Escherichia coli↗
PubMed1989

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes.

Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.

Acetyltransferases↗
PubMed1985

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.

Affinity Labels↗