Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

Flexibility and folding of phosphoglycerate kinase

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.     

Microdistribution and local dosimetry of 226Ra in trabecular bone of the beagle

Sections of lumbar vertebral bodies of young adult beagle dogs have been analyzed autoradiographically to characterize and quantify the local distribution of 226Ra by means of a scanning microscope photometer. The animals received a single injection of 355 kBq/kg body weight and were serially sacrificed at 5 to 1381 days postinjection. Hotspot concentrations decreased from about 51 kBq/g bone at 5 days to 20 kBq/g at 1381 days postinjection. The diffuse concentration changed from 8.3 to 1.9 kBq/g. The mean 226Ra concentration in the trabecular areas scanned was initially higher and at the end of the observation period lower than the average calculated for the whole lumbar vertebral column. Density and area of, and fraction of bone activity in, hotspots virtually remained constant. With time hotspots tended to become translocated into bone volume. Mean dose rates to lining cells from both hotspots and diffuse labels decreased from about 210 mGy/d at early postinjection times to 105 mGy/d. This corresponds to 2.5 to 1.1 times the average skeletal dose rate. A discussion of the level of irradiation in terms of hit frequencies shows that osteoblasts in the initial phase of hotspot formation receive about 60 hits to their nucleus for the duration of bone formation. After about 6 months, however, the 226Ra concentration in new bone and the corresponding hit frequency appears to be low enough that interference with bone formation is unlikely. Morphometric measurements showed that abnormal bone accretion and thickening of trabeculae occurred. This was interpreted as an imbalance between bone formation and resorption. Both formation and resorption seem to be substantially lowered compared to control animals.     

Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.     

Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these different data and the thermodynamic analysis, it was suggested that the two domains of the horse muscle phosphoglycerate kinase refold independently of one another with different equilibrium constants, the most favorable constant referring to the folding of the C-terminal domain which contains all tryptophans.     

Enzyme purification by hydrophobic chromatography: an alternative approach illustrated in the purification of aspartate transcarbamoylase from wheat germ (Short Communication)

Two adsorbents containing similar numbers of hydrocarbon (C(10)) chains but different numbers of carboxyl groups were made by chemical modification of Sepharose. The use of these adsorbents to purify proteins, under conditions where hydrophobic adsorption is partly resisted by electrostatic repulsion, is illustrated in the purification of aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ.     

Cloning and sequencing of the gerD gene of Bacillus subtilis

A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsI. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnI. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1.8 kb fragment of DNA which complemented ger-97::Tn917 and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential sigma E and sigma A promoter regions and encoding a predicted polypeptide of 21,117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.     

The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis.

The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min-1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min-1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.