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Biomedical subjects

M L Hackert

Publications and source records attributed to M L Hackert.

47 records · Page 3Linked to original sources
PubMed1977

Purification and crystallization of NADP+-specific isocitrate dehydrogenase from Escherichia coli using polyethylene glycol.

A simple and rapid method is presented for purifying the NADP+-dependent isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), from Escherichia coli, which relies on fractionation of the enzyme with polyethylene glycol. The shortened preparation results in a 32% relative recovery of purified enzyme at a specific activity of 127 micronmol/min per mg of protein. The Km values for threo-DS-isocitrate, NADP+, NAD+, Mg2+ and Mn2+ are 6.4, 36, 3000, 19.7 and 2.0 micronM, respectively. The stability of the enzyme as a function of dilution and temperature are also reported. Recrystallization of the purified enzyme under different conditions readily produces a variety of single crystals. Crystals grown from ammonium sulfate solutions belong to monoclinic space group C2 with a = 125 A, b = 111 A, c = 83.5 A and beta = 108degrees 45'. Density measurements of these crystals indicate there are two 80 000-dalton dimers per asymmetric unit.

Crystallization↗
PubMed1973

Structure-function relationships in lactate dehydrogenase.

The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate.

Amino Acid Sequence↗
PubMedDate not supplied

Evolution of cooperativity in hemoglobins: what can invertebrate hemoglobins tell us?

While vertebrate hemoglobins typically are tetrameric and show highly regulated and cooperative ligand binding, little is known of the evolution of these properties. We are studying the structural and functional properties of the hemoglobins from Caudina arenicola, an echinoderm. The echinoderms are in the lineage most closely related to the vertebrates to express hemoglobin. C. arenicola has three sets of red cells, in the water vascular system, the coelomic cavity, and in an intestinal vein. Each of these expresses a distinct array of globins. The hemoglobins are cooperative and exhibit unusual ligand-linked associative properties, being dimeric when oxygenated and forming tetramers and higher aggregates on deoxygenation. The major coelomic hemoglobins have been subjected to a detailed examination by a combination of ligand binding analyses and protein and DNA sequencing, as well as X-ray crystallography. Two typical globin introns were identified, along with a unique intron that bisects an N-terminal extension of the globin from the remainder of the gene. X-ray crystallographic analysis shows that the subunit interfaces of C. arenicola hemoglobins differ radically from those of vertebrate hemoglobins and indeed from some other invertebrate hemoglobins, but closely resemble the packing arrangements found in a clam hemoglobin (Scapharca). However, the residues implicated in cooperativity in these two types of hemoglobins differ substantially.

Animals↗