This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk ␣-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of ␣-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule ␣-lactalbumin showed that the active fraction contains oligomers of ␣-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve ␣-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of ␣-lactalbumin.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBDI) and the regulatory domain (R). Tryptic digests of NBDI-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 1 18 species) containing Ser residues within "high-probability'' PKA consensus sequences: R-R/K-X-SIT, R-X-X-SIT, and R-X-S/T. Three criteria were used to assign phosphorylated sites: ( I ) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; ( 2 ) co-elution with the PO,-ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBDl and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser4", Ser5I9, Ser670, and Thr7", were not phosphorylated. The "lowprobability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.

CHIP28 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP28 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP28. In purified native CHIP28 from erythrocytes, approximately 50% of CHIP28 molecules were glycosylated; each mole of glycosylated CHIP28 contained 5.4 kDa of monosaccharides consisting of 2 mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP28 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP28 remained tightly associated when solubilized in octyl beta-D-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP28 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP28 eluted as an apparent dimer, whereas deglycosylated CHIP28 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP28 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.2 x 10(-14) cm3/s for native CHIP28. Circular dichroism of native and deglycosylated CHIP28 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP28 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Dry powder inhalers (DPIs) are increasingly being used for the treatment of asthma and COPD. A potential drawback is that DPIs can be sensitive to humidity. Two DPIs, Symbicort Turbuhaler and Seretide Diskus, were stored 3 months at either 25 degrees C/30% RH or 40 degrees C/75% RH. After storage, delivered, as well as fine particle dose, FPD, were tested in vitro and lung deposition, of the steroid components, was assessed in vivo. After storage at 40 degrees C/75% RH, delivered dose as well as FPD from Symbicort Turbuhaler was virtually unchanged while FPD for Seretide Diskus decreased by about 50% despite no decrease in delivered dose. For both products, no difference in FPD was seen after storage at 25 degrees C/30% RH. These in vitro findings were confirmed in the in vivo part of the study. Lung deposition for Symbicort Turbuhaler was unaffected by 40 degrees C/75% RH storage, while for Seretide Diskus it was reduced with about 50%. The study extends previous in vitro observations of impaired performance of Seretide Diskus and demonstrates that this translates into decreased drug delivery to the site of action. The clinical importance of this finding has not been studied but could result in undertreatment.

The gastric H,K-ATPase is responsible for acid secretion by parietal cells. Its beta-subunit is a glycoprotein which is exposed to the harsh, acidic environment of the stomach. The location and structural features of the N-linked oligosaccharides were determined using matrix-assisted laser desorption ionization mass spectrometry (MALDI/MS) (in conjunction with mass composition analysis and exoglycosidase digestions), Edman degradation, and monosaccharide composition analysis. All seven N-linked sequons at positions 99, 103, 130, 146, 161, 193, and 222 were fully glycosylated. An unusual restricted array of oligosaccharides was observed at individual Asn residues. Asn99 was modified exclusively with oligomannosidic-type structures (Man6GlcNAc2-Man8GlcNAc2). Asn193 contained both oligomannosidic (Man5GlcNAc2-Man8GlcNAc2) and lactosamine-type structures, indicating significant "leakiness" in the pathway which converts oligomannose to lactosamine-type at a single glycosylation site. MALDI/MS with collision-induced dissociation was required to demonstrate that sequons separated by a single residue (99Asn-Ile-Ser-Asp-Asn-Arg-Thr105) were modified with only oligomannose and lactosamine structures, respectively. Analysis of the total oligosaccharide pool using MALDI/MS and exoglycosidase analysis revealed 24 lactosamine species (bi-, tri-, and tetraantennary structures), with all branches terminated in alpha-linked Gal residues, most possessing a single Fuc residue. Nine novel oligosaccharides contained multiple alpha-linked Gal residues per branch. Bi- and triantennary structures, with and without lactosamine repeats, were observed at Asn146 and Asn161. Tetraantennary structures with lactosamine repeats were found only at Asn130, and this site also contained most of the structures with multiple alpha-linked Gal residues per branch.