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Biomedical subjects

L M Keefer

Publications and source records attributed to L M Keefer.

11 recordsLinked to original sources
PubMed1988

Adenylate cyclase and beta-receptors in salivary glands of rats fed diets containing trans fatty acids.

The effects of trans fatty acids on adenylate cyclase were determined in the submandibular salivary glands (SMSG) of rats fed diets containing 20% corn oil, 20% partially hydrogenated soybean oil (PHSBO) or 18% PHSBO + 2% corn oil. The fluoride- and isoproterenol-stimulated adenylate cyclase activities were higher in the SMSG from rats fed 20% PHSBO than in the control group fed 20% corn oil. The feeding of 2% corn oil with the diet containing 18% PHSBO resulted in a complete restoration of isoproterenol-stimulated and a partial restoration of fluoride-stimulated adenylate cyclase activity. There was no significant difference in the concentration of the beta-adrenergic receptor or in the receptor-binding affinity constants among the three dietary groups as measured by [3H]dihydroalprenolol (DHA)-binding. Higher fluorescence polarization of diphenyl hexatriene (DPH) was observed in SMSG membranes of rats fed diet containing 20% PHSBO than in the other two oils, suggesting that membrane fluidity may play a role in adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1987

Hepatic prolactin binding is rapidly altered by endotoxin in lactating mice.

Endotoxin or lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, produces profound physiologic changes in most mammals. The effects of LPS on ovine prolactin (oPRL) binding by hepatic membranes of lactating mice is explored in this report. Specific 125I-oPRL binding by liver membranes from LPS-responder C3HfB/HeN mice increased two-fold within fifteen minutes of the injection of LPS, while no change was observed in the non-responder C3H/HeJ mice. Specific 125I-insulin binding did not change. Scatchard analysis of equilibrium binding of oPRL to C3HfB/HeN liver membranes indicated that within fifteen minutes of LPS injection, a receptor of differing binding affinity appears and then disappears by one hour post-injection. We propose that these rapid alterations in the specific binding of oPRL by liver membranes from LPS-injected, lactating C3HfB/HeN mice are due to the transient creation or unmasking of a novel class of PRL receptor.

Animals↗
PubMed1984

Monoclonal antibodies and specific cell surface receptors do not discriminate between human growth hormone prepared by DNA recombinant techniques and the native hormone.

Native and E. coli-derived human growth hormone (hGH) have been compared in their ability to react with four different monoclonal antibodies (MAbs) as well as with the specific cell surface receptors of cultured human IM-9 lymphocytes and liver membranes of pregnant rabbit. For all the tests used, the competition curves obtained using both hormone preparations were superimposable. The results suggest a close similarity in the immunological and biological (binding reaction) properties of native and recombinant hGH.

Antibodies, Monoclonal↗
PubMed1981

Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120.

A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described. The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits. Sequence analysis suggests that the subunits are identical. There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli. NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources. The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E. coli.

Amino Acid Sequence↗
PubMed1981

Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors.

Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells. The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors. Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells. We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B. Frank, Eli Lilly Research Laboratories). In all aspects studied, the pure [TyrA14-125I]iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration.

Cells, Cultured↗
PubMed1978

D-3-Phosphoglycerate dehydrogenase from chicken liver. I. Purification.

A method is described for the preparation of homogeneous D-3-phosphoglycerate dehydrogenase from chicken liver in amounts sufficient for structural studies. The procedure utilizes ammonium sulfate precipitation, blue dextran-Sepharose chromatography, ion exchange chromatography on phosphocellulose, and crystallization. Previous reports of instability of the enzyme have been shown to be due to proteolysis in the crude extract which can be effectively prevented by leupeptin. The purified enzyme is a basic protein with a pI of 8.95 as measured by isoelectric focusing. The extinction coefficient at 278 nm of a 1% solution is 5.3.

Animals↗
PubMed1977

Structural studies on Halobacterium halobium bacteriorhodopsin.

Bacteriorhodopsin, isolated from Halobacterium halobium, is an intrinsic membrane protein containing covalently attached retinal and driven by a cyclic light-driven protonmotive force. In order to extend three-dimensional analyses already in progress and to define the covalent features of such proteins, the determination of the primary structure of bacteriorhodopsin has been initiated, using techniques that can be applied to the sequence analyses of other intrinsic membrane proteins as well. The amino acid composition of bacteriorhodopsin prepared by new procedures compares well to that previously published. Various delipidation procedures have been examined for their effectiveness in yielding protein in a physical state that is susceptible to fragmentation by a number of chemical and proteolytic agents. Organic extraction, particularly in acetone:ammonium hydroxide, has proved most useful. Detergent extraction was effective in delipidating but rendered the protein inert to attack by various cleavage reagents. Sequence information was obtained from fragments isolated from several hydrolytic preparations of low percent cleavage. The most complete fragmentation was obtained from hydrolysis of bacteriorhodopsin previously modified with succinic or maleic anhydride. Both solubility and fragmentation susceptibility were enhanced by these reactions and several other derivatives of bacteriorhodopsin are currently being investigated. The behavior of bacteriorhodopsin under the conditions examined, as well as the sequence information obtained, suggest that bacteriorhodopsin is mostly contained within the lipid milieu of the membrane and may represent a class of membrane proteins that do not contain appreciable hydrophilic domains.

Amino Acids↗
PubMedDate not supplied

Receptor binding properties and biologic activity in vitro of biosynthetic human insulin.

The interaction of biosynthetic human insulin with human cultured lymphocytes, human circulating erythrocytes, and isolated rat fat cells was examined. The binding of the biosynthetic insulin to human cells was identical to that of native human or pork insulins, with respect to affinity, kinetics of association and dissociation, negative cooperativity, and downregulation of lymphocyte receptors. The biosynthetic insulin also had equal potency in stimulating the incorporation of [3H]-glucose into the lipids of isolated rat fat cells. These data suggest that the structure of the biosynthetic insulin has been integrally reconstituted.

Adipose Tissue↗