Sigmoidal kinetics of glucokinase.

Glucokinases obtained from the liver of several species of mammals and amphibians exhibit sigmoidal saturation functions for glucose. Hill coefficients (nH) are about 1.5, and half-saturation values (K0.5) lie between 1.5 and 8.5 mmol/l. The nH and K0.5 values are constant throughout the purification steps of rat glucokinase. A dimeric form of rat glucokinase appearing in aged preparations exhibits michaelian kinetics. Sigmoidal kinetics is considered as an adaptive feature of glucokinases to increase the efficiency of the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.     

Phylogenetic relationships of bolitoglossine salamanders: a demonstration of the effects of combining morphological and molecular data sets

We analyzed sequence data for 555 bp of the mitochondrial gene cytochrome b in plethodontid salamanders, taken from 18 ingroup (tribe Bolitoglossini) and 4 outgroup (tribe Plethodontini) taxa. There were 257 variable sites, of which 219 were phylogenetically informative. Sequence differences among taxa exceeded 20%, and there were up to 15% amino acid differences among the sequences. We also analyzed 37 morphological (including karyological) characters, taken from the literature. Data were analyzed separately and then combined using parsimony and likelihood approaches. There is little conflict between the morphological and DNA data, and that which occurs is at nodes that are weakly supported by one or both of the data sets. Treated separately, the morphological and DNA data provide strong support for some nodes but not for others. The combined data act synergistically so that good support is obtained for nearly all of the nodes in the tree. Recent divergences are supported by silent transitions, and older divergences are supported by a combination of morphological, karyological, DNA transversion, and amino acid changes. Eliminating silent changes from the DNA data improves the consistency index and improves some bootstrap and decay index values for several deeper branches in the tree. However, the combined data set with all characters included provides a better supported tree overall. Maximum likelihood and parsimony with all of the data give not only the same topology but also remarkably similar branch lengths. Results of this analysis support the monopoly of the supergenera Hydromantes and Batrachoseps, and of a sister group relationship of Batrachoseps and the supergenus Bolitoglossa (represented in this study one species of the genus Bolitoglossa).      

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.     

Probing protein conformation with a minimal photochemical reagent

3H-diazirine (3H-DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3H-DZN generates 3H-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine alpha-lactalbumin (alpha-LA, MW: 14,200) with 1 mM (3)H-DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of alpha-LA, as monitored by the extent of 3H-methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25-30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the approximately 100% increment expected from theoretical estimates of ASA of state U. Among other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the alpha subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the 3H-methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of nonnative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.     

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

Background  

The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors.     

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and −0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.     

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.     

Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units per oocyte in the absence of glucose-6-P. V(max) in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 microM and, depending on substrate concentrations, 50-100 microM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.