Partial assignment of disulfide pairs in neurophysins

The original report assigning the pairing of neurophysin's 14 half-cystine residues (Schlesinger et al. (1972), Proc. Natl. Acad. Sci., U.S.A., 69,3350-3353) was based on an incorrect amino acid sequence. In the present study, re-investigation of the results of proteolytic fragmentation of bovine neurophysins indicates that the majority of the original assignments were incorrect. Three disulfide pairs are now assigned as Cys21-Cys44, Cys67-Cys85 and Cys74-Cys79. The pairing pattern indicates that neurophysin's variable carboxyl terminal region, separately encoded by the third gene exon, does not form a self-contained domain.     

A mass spectrometric technique for detecting and identifying by-products in the synthesis of peptides

The utility of a new mass spectrometric technique for detecting and identifying peptide by-products produced in the synthesis of peptides is demonstrated. The technique involves three sequential steps: (1) practically nondestructive 252Cf plasma desorption mass spectrometric analysis of monolayer amounts of the peptide(s) of interest bound to a thin layer of nitrocellulose; (2) enzyme-catalyzed microscale chemical reaction of the surface-bound peptide(s) to produce structurally informative hydrolysis products; (3) plasma desorption mass spectrometric analysis of these hydrolysis products. The first step determines the presence and the molecular weights of unwanted by-products resulting from errors or incomplete reactions during synthesis. The subsequent two steps provide information on the precise location in the peptides where errors have occurred. In the present paper, the technique is applied to an investigation of unwanted peptide by-products associated with the use of tryptophan during stepwise solid-phase peptide synthesis. Synthetic preparations of melittin and [Bpa-8]dynorphin A (1-17) were each found to contain a major impurity with molecular weight 28 Da higher than that of the desired product. The impurity in the melittin preparation, in which the final deprotection step involved the high-low HF procedure, was shown to result from incomplete removal of the formyl group from Trp-19. On the other hand, the impurity in the [Bpa-8]dynorphin A (1-17) preparation, where the removal of the formyl group from Trp-14 was carried out using piperidine, was shown to result from migration of the formyl group to Lys-11 or Lys-13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Secretion of a lipid transfer protein by human monocyte-derived macrophages

Human monocyte-derived macrophages in culture were shown to synthesize and secrete a lipid transfer protein. The human monocyte-derived macrophage transfer protein showed the following characteristics: (i) linear secretion rate over a 24-h period, which was blocked completely by cycloheximide and stimulated by phorbol myristate acetate (67% increase over nonstimulated values); (ii) apparent Mr = approximately 62,000 off Sephacryl S-200; (iii) isoelectric point of 5.0; (iv) binding to phenyl-Sepharose, but not to heparin-Sepharose; (v) facilitation of the transfer of both neutral lipids (cholesteryl esters and triglycerides) and phosphatidylcholine between high density lipoproteins and d less than 1.063 g/ml lipoproteins; and (vi) thermal stability (stable for 1 h at 56 degrees C). The last five of these properties are similar to those of the plasma lipid transfer protein. Thus, macrophages secrete a lipid transfer protein that closely resembles the neutral lipid transfer protein found in human plasma and may be a source of this plasma protein in vivo.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.