Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Purification of duck growth hormone and cloning of the complementary DNA

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Is the insulin resistance of patients with noninsulin-dependent diabetes mellitus secondary to insulin deficiency?

Defects in both insulin secretion and action have been documented in patients with noninsulin-dependent diabetes mellitus (NIDDM), leading to the suggestion that both fasting hyperglycemia and insulin resistance in NIDDM are secondary to insulin deficiency. In order to test this hypothesis, insulin secretion (plasma insulin response to oral glucose) and insulin action (insulin clamp) were determined in 25 patients with NIDDM. The results documented relationships between incremental plasma insulin response to glucose and degree of fasting hyperglycemia (r = -.045, P less than 0.05) and insulin-stimulated glucose utilization (r = 0.25, P = NS). These data indicate that differences in insulin secretory response accounted for only approximately 20% of the variance in fasting plasma glucose level and 6% of the variance in insulin resistance in NIDDM. Thus, differences in insulin-secretory response contribute modestly to magnitude of glycemia, and not at all to variations in insulin resistance in NIDDM, permitting rejection of the hypothesis that insulin resistance is secondary to insulin deficiency.     

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Co-operativity and enzymatic activity in polymer-activated enzymes. A one-dimensional piggy-back binding model and its application to the DNA-dependent ATPase of DNA gyrase

The binding of a ligand to a one-dimensional lattice in the presence of a second ("rider") ligand, which binds only to the first ligand (piggy-back binding), is studied. A model derived from this study is used to analyze the effects of co-operativity on the reaction rates of enzymes activated by polymeric cofactors that provide multiple binding sites for the enzyme. It is found that in the presence of strong co-operativity, the steady-state reaction rates of polymer-activated enzymes can be very different from the Michaelis-Menten paradigm. By adjusting the co-operativity parameters and the binding constants of the ligands, the model can generate apparent auto-catalytic enhancement by substrates at low substrate concentrations and apparent substrate inhibition at high substrate concentrations. The model is shown to be able to explain the differences in the rates of ATP hydrolysis by DNA gyrase in the presence of long versus short DNA molecules and in the presence of long DNA molecules at different gyrase to DNA ratios.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Identification of the pleiotropic sacQ gene of Bacillus subtilis.

The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.     

Design of a system for the control of low dissolved oxygen concentrations: critical oxygen concentrations for Azotobacter vinelandii and Escherichia coli

The physiological activity of microorganisms in environments with low dissolved oxygen concentrations often differs from the metabolic activity of the same cells growing under fully aerobic or anaerobic conditions. This article describes a laboratory-scale system for the control of dissolved oxygen at low levels while maintaining other parameters, such as agitator speed, gas flowrate, position of sparger outlet, and temperature at fixed values. Thus, it is possible to attribute in dilute nonviscous fermentations all physiologic changes solely to changes in dissolved oxygen. Experiments were conducted with Azotobacter vinelandii and Escherichia coli. Critical oxygen concentrations for growth (that value of oxygen allowing growth at 97% of mu max) were measured as 0.35 +/- 0.03 mg/L for A. vinelandii and 0.12 +/- 0.03 mg/L for E. coli. These values are significantly different from the commonly quoted values for critical oxygen concentrations based on respiration rates. Because of the superior dissolved oxygen control system and an improved experimental protocol preventing CO2 limitation, we believe that the values reported in this work more closely represent reality.