Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor

Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Spin trap determination of free radical burst kinetics in stimulated neutrophils

Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.     

Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells

Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.     

A proton and carbon 13 nuclear magnetic resonance study of neomycin B and its interactions with phosphatidylinositol 4,5-bisphosphate

The entire proton NMR spectrum of the aminoglycoside antibiotic neomycin B has been assigned at physiological pH by a combination of two-dimensional J-resolved and J-correlated and nuclear Overhauser enhancement difference spectroscopy. Unambiguous assignment of all four ring systems is possible without recourse to model or derivative compounds by observing nuclear Overhauser enhancements between as well as within rings. The subsequent assignment of the carbon 13 spectrum is simply achieved using two-dimensional heteronuclear J-correlated techniques. The proton NMR spectrum of a sonicated aqueous dispersion of the intracellular second messenger precursor phosphatidylinositol 4,5-bisphosphate is reported for the first time. The spectrum is consistent with a high degree of side chain unsaturation and a conformation for the myo-inositol head group, which appears highly mobile, in which all bulky substituents are equatorial (except the 2-hydroxyl). Addition of aliquots of phosphatidylinositol 4,5-bisphosphate to an aqueous buffered solution of neomycin B induces complex changes in the whole spectrum of the latter, including downfield shifts of differential magnitude for several well-resolved signals, viz. the anomerics, and the pair of methylene protons of the substituted cyclohexane. The complexation kinetics are fast on the NMR time scale at 25 degrees C. The binding results are discussed in terms of a tentative complexation geometry.     

The effect of 2,3-diphosphoglycerate on the tetramer-dimer equilibrium of carbon monoxide hemoglobin in dilute solution. Correlation between sedimentation and kinetic behavior

The effect of 2,3-diphospho-D-glycerate on the sedimentation coefficient of carbon monoxide hemoglobin was correlated with the fraction of rapidly reacting hemoglobin observed subsequent to flash photolysis at 23 degrees C at pH 7.30 in buffers of 0.1 M ionic strength. Concentrations of the organic phosphate up to about 5 mM resulted in an increase in S20,w, consistent with an increase in the fraction of tetrameric hemoglobin. A decrease in rapidly reacting hemoglobin parallelled the increase in the sedimentation coefficient. Between 5 and 20 mM 2,3-diphosphoglycerate, S20,w decreased, suggesting that dissociation to dimers was enhanced. An increase in rapidly reacting hemoglobin was also observed in this concentration range. Similar sedimentation results were obtained with oxyhemoglobin at pH 7.00 and carbon monoxide hemoglobin at pH 7.06. Assuming single binding sites on each species, the dissociation constants for 2,3-diphosphoglycerate binding to tetrameric and dimeric HbCO are 0.2-0.3 mM and 2-5 mM at pH 7.30. This biphasic effect of this physiologically important organic phosphate on the state of aggregation of R state hemoglobin has not been previously reported, but it is similar to that previously noted with inositol hexaphosphate, which enhanced tetramer formation at low concentrations, while at higher concentrations it promoted hemoglobin dissociation to dimers (White, S. L. (1976) J. Biol. Chem. 251, 4763-4769; Gray, R. D. (1980) J. Biol. Chem. 255, 1812-1818).     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.