Unusually weak oxygen binding, physical properties, partial sequence, autoxidation rate and a potential phosphorylation site of beluga whale (Delphinapterus leucas) myoglobin.

We purified myoglobin from beluga whale (Delphinapterus leucas) muscle (longissimus dorsi) with size exclusion and cation exchange chromatographies. The molecular mass was determined by mass spectrometry (17,081 Da) and the isoelectric pH (9.4) by capillary isoelectric focusing. The near-complete amino acid sequence was determined and a phylogeny indicated that beluga was in the same clad as Dall's and harbor porpoises. There were consensus motifs for a phosphorylation site on the protein surface with the most likely site at serine-117. This motif was common to all cetacean myoglobins examined. Two oxygen-binding studies at 37 degrees C indicated dissociation constants (20.5 and 23.6 microM) 5.7-6.6 times larger than horse myoglobin (3.6 microM). The autoxidation rate of beluga myoglobin at 37 degrees C, pH 7.2 was 0.218+/-0.028 h(-1), 1/3 larger than reported for myoglobin of terrestrial mammals. There was no clear sequence change to explain the difference in oxygen binding or autoxidation although substitutions (N66 and T67) in an invariant rich sequence (HGNTV) distal to the heme may play a role. Structural models based on the protein sequence and constructed on topologies of known templates (horse and sperm whale crystal structures) were not adequate to assess perturbation of the heme pocket.

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.

Identification of collagen-binding proteins in Lactobacillus spp. with surface-enhanced laser desorption/ionization-time of flight ProteinChip technology.

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)-time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.

Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes.

The reversible acetylation of histones is associated with structural alterations in the chromatin fiber that affect various DNA-related activities. Here we show that the histone acetyltransferase p300 specifically acetylates HMG-14, a nonhistone structural protein that binds to nucleosomes and reduces the compactness of the chromatin fiber. We identify 7 major acetylation sites, 6 of which are novel and have not been known to be acetylated in either HMG-14 or the closely related HMG-17 protein. All the acetylation sites involve evolutionarily conserved residues: 3 within the HMG-14/-17 nucleosomal binding domain and 4 in or near the bipartite nuclear localization domains of the proteins. In tissue culture cells the acetylation pattern is indicative of a selective process in which a subfraction of HMG-14 is preferentially acetylated. We find that the nucleosomal binding domain is a major target for acetylation in vivo and that the specific acetylation of HMG-14 by p300 weakens its interaction with nucleosome cores. Our results suggest that p300 modulates the interaction of HMG-14 with nucleosomes. Thus, p300 may affect chromatin-related activities not only by modifying histones or transcription factors but also by targeting structural nonhistone proteins.

Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress.

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl2 and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO2 treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels.

Gustin from human parotid saliva is carbonic anhydrase VI.

Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previously isolated and characterized as a single polypeptide chain of 37kDa with one mole of zinc tightly bound to the protein. It exhibited biological activity activating calmodulin dependent bovine brain cAMP phosphodiesterase and was decreased in saliva of patients with loss of taste in whom taste buds showed a specific pathological morphology. Determination of its primary structure by amino acid sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reverse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37kDa, the deglycosylated protein, 33kDa. N-linked carbohydrate chains contained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five increasingly acidic pI values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked glycoforms of the protein. Gustin was found to exhibit CA activity but was inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigators, indicate that parotid saliva gustin is CA VI.