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K F Faull

Publications and source records attributed to K F Faull.

At least 55 records · Page 3Linked to original sources
PubMed1998

Porcine cerebroside sulfate activator (saposin B) secondary structure: CD, FTIR, and NMR studies.

Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical-chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of alpha-helical conformation and little if any beta-sheet. Circular dichroism (CD) studies indicate 45-50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85 degrees C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.

Algorithms↗
PubMed1998

Revised primary structures of rat pituitary gamma-lipotrophin and beta-endorphin.

In-depth investigations, by high performance liquid chromatographic purification, radio-immunoassay, mass spectrometry, tandem mass spectrometry, Edman sequencing and limited C-terminal ladder sequencing, were prompted by mass spectrometric charting experiments which suggested that the amino acid sequences for rat gamma-lipotrophin and beta-endorphin require revision. The results for gamma-lipotrophin identify a histidine for glutamine substitution at position 12, and heterogeneity in the expressed protein presumably due to partial dehydration. Partial dehydration for acidic joining peptide, previously reported by Toney et al was corroborated. The results for beta-endorphin confirm the presence of alanine at position 26 and provide no evidence for the expression of multiple forms of the hormone.

Amino Acid Sequence↗
PubMed1997

Carbohydrate structures of recombinant human alpha-L-iduronidase secreted by Chinese hamster ovary cells.

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant alpha-L-iduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.

Amino Acid Sequence↗
PubMed1997

Anchor structure of staphylococcal surface proteins. A branched peptide that links the carboxyl terminus of proteins to the cell wall.

Surface proteins of Staphylococcus aureus are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal. Previous work demonstrated that the sorting signal is cleaved at the conserved LPXTG motif and that the carboxyl of threonine (T) is linked to the staphylococcal cell wall. By employing different cell wall lytic enzymes, surface proteins were released from the staphylococcal peptidoglycan and their COOH-terminal anchor structure was revealed by a combination of mass spectrometry and chemical analysis. The results demonstrate that surface proteins are linked to a branched peptide (NH2-Ala-gamma-Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the pentaglycine cross-bridge that is attached to the epsilon-amino of lysyl. This branched anchor peptide is amide-linked to the carboxyl of N-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.

Amidohydrolases↗
PubMed1997

Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo.

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

Animals↗
PubMed1997

Purification, amino acid sequence, synthesis, and receptor selectivity of alligator gastrin.

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.

Alligators and Crocodiles↗
PubMed1997

Monocyte binding to endothelial cells induced by oxidized phospholipids present in minimally oxidized low density lipoprotein is inhibited by a platelet activating factor receptor antagonist.

Our group has previously demonstrated that oxidized phospholipids derived from mildly oxidized low density lipoprotein (MM-LDL) or oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) increase monocyte-endothelial interactions. There are indications that the effects of these phospholipids are receptor mediated. Oxidized phospholipids have previously been shown to activate cells via the platelet activating factor (PAF) receptor, therefore, we pretreated human aortic endothelial cells (HAECs) with a PAF-receptor antagonist (WEB 2086) prior to treatment with oxPAPC. WEB 2086 inhibited monocyte binding to endothelial cells induced by oxPAPC (200 micrograms/ml) at concentrations from 1 nM to 10 microM, but had no effect on the induction of monocyte binding induced by lipopolysaccharide (LPS). We were able to isolate and identify several active oxidized phospholipids by combined normal phase high performance liquid chromatography (HPLC) and electrospray mass spectrometry (LC/MS). The induction of monocyte binding to HAECs by two of these partially purified phospholipids was totally abolished by the pretreatment of HAECs with WEB 2086 (100 nM). PAF itself, however, when tested at concentrations from 10 nM to 10 microM had no effect on monocyte binding. These results suggest that several of the oxidized phospholipids present in MM-LDL and oxPAPC induce monocyte binding through a receptor which is perhaps distinct from the PAF-receptor, but can be blocked by the PAF-receptor antagonist WEB 2086.

Aorta↗
PubMed1995

Neuroamine related compounds in the CSF of hydrocephalic rabbits.

The effects of neonatal hydrocephalus on the levels of tyrosine, tryptophan, 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in CSF were determined by high-performance liquid chromatography (HPLC) with fluorometric detection in normal and chronically hydrocephalic rabbits. The hydrocephalic rabbits showed a highly significant increase in both the serotonin metabolite 5-HIAA and the dopamine metabolite HVA. There were no significant effects of the hydrocephalus on either tyrosine or tryptophan levels. There was a significant positive correlation between the intracranial pressure (ICP) and the increase in 5-HIAA and HVA, but not with the two precursor amino acids. There was a significant decrease in these amino acid precursors with age in both groups. A trend towards higher levels of 5-HIAA and HVA in older rabbits was also evident, however this change was not to the degree found in the hydrocephalics. These data indicate that increased ICP affects the mechanism of removal of 5-HIAA and HVA from the cerebrospinal fluid.

Aging↗
PubMed1995

3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae.

The biosynthesis of ubiquinone (coenzyme Q) was studied in Saccharomyces cerevisiae. Lipid extracts were prepared from wild-type yeast grown in the presence of p-[U-14C]- and p-[carboxy-14C]hydroxybenzoic acid. Ergosterol was removed by adsorption to digitonin-celite, and radiolabeled lipids were purified by sequential reverse-phase and normal-phase HPLC steps. Radiolabeled peaks were identified by comparison with synthetic standards using retention time and electron ionization mass spectrometric criteria. The recovery and identification of the unstable 3-hexaprenyl-4-hydroxybenzoic acid molecule were facilitated by treatment of the lipid extract with diazomethane under conditions that resulted in the formation of the stable derivatives methyl 3-hexaprenyl-4-hydroxybenzoate or methyl 3-hexaprenyl-4-methoxybenzoate. In stationary-phase yeast cultures, the major radioactive lipid products are coenzyme Q and 3-hexaprenyl-4-hydroxybenzoic acid, constituting 62 and 38% of the radioactive lipids, respectively. However, under log-phase growth conditions the biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid predominates (accounting for 81% of the radioactive lipids). The data indicate that in wild-type yeast, 3-hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis and that in log-phase growth this ubiquinone intermediate is present at fourfold higher abundance than the end product. The physiological rationale for this high concentration of a membrane-bound intermediate is unclear.

Benzoates↗
PubMed1995

Structure of the cell wall anchor of surface proteins in Staphylococcus aureus.

Many surface proteins are anchored to the cell wall of Gram-positive bacteria and are involved in the pathogenesis of these organisms. A hybrid molecule was designed that, when expressed in Staphylococcus aureus, was anchored to the cell wall and could be released by controlled enzymatic digestion. By a combination of molecular biology and mass spectrometry techniques, the structure of the cell wall anchor of surface proteins in S. aureus was revealed. After cleavage of surface proteins between threonine and glycine of the conserved LPXTG motif, the carboxyl of threonine is amide-linked to the free amino group of the pentaglycine crossbridge in the staphylococcal cell wall.

Amino Acid Sequence↗
PubMed1995

In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation.

Early stages of nodulation involve the exchange of signals between the bacterium and the host plant. Bacterial nodulation (nod) genes are required for Rhizobium spp. to synthesize lipooligosaccharide morphogens, termed Nod factors. The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products. Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction. Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification. NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.

Bacterial Proteins↗
PubMed1995

Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein.

Our group has previously demonstrated that oxidized phospholipids in mildly oxidized LDL (MM-LDL) produced by oxidation with lipoxygenase, iron, or cocultures of artery wall cells increase monocyte-endothelial interactions and this sequence of events is blocked by HDL. To obtain further insight into the mechanism by which HDL abolishes the activity of MM-LDL we investigated the effect of the HDL-associated ester hydrolase paraoxonase (PON). Treatment of MM-LDL with purified PON significantly reduced the ability of MM-LDL to induce monocyte-endothelial interactions. Inactivation of PON by pretreating HDL with heat or EDTA reduced the ability of HDL to inhibit LDL modification. HPLC analysis of phospholipids isolated from MM-LDL before and after treatment with purified PON showed that the 270 nm absorbance of phospholipids was decreased, while no effect was observed on 235 nm absorbance. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and specific fractions of Ox-PAPC isolated by HPLC induced the same monocyte-endothelial interactions as did MM-LDL. Biologically active and inactive HPLC fractions of Ox-PAPC were compared by fast atom bombardment-mass spectrometry which revealed that active fractions possessed ions with a mass to charge [correction of change] ratio greater than native PAPC by multiples of 16 D suggesting the addition of 3 and 4 oxygen atoms to PAPC. Comparison of Ox-PAPC by fast atom bombardment-mass spectrometry before and after PON treatment showed that PON destroyed these multi-oxygenated molecules found in biologically active fractions of Ox-PAPC. These results suggest that PON in HDL may protect against the induction of inflammatory responses in artery wall cells by destroying biologically active lipids in mildly oxidized LDL.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1995

Tear lipocalins bind a broad array of lipid ligands.

To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

Carrier Proteins↗
PubMed1994

Effects of raclopride treatment on plasma and CSF HVA: relationships with clinical improvement in male schizophrenics.

Thirty-two acutely psychotic, male schizophrenic patients received raclopride, at 2, 6, or 12 mg/day, or haloperidol, 15 mg/day for 4 weeks after randomized, double-blind assignment. Twenty-six patients, including 19 who had been assigned one of the three doses of raclopride, completed the study. Raclopride, particularly at 12 mg/day, increased CSF homovanillic acid (HVA) at 4 weeks, and plasma HVA at 2 days, of treatment. The clinical response to raclopride was significantly correlated with plasma raclopride concentrations and baseline plasma HVA concentrations. Although raclopride is a substituted benzamide with atypical properties in animals, these results suggest that the doses of raclopride required for clinical efficacy and elevation of clinical indices of brain dopamine turnover are similar.

Adult↗
PubMed1994

The effect of chronic alprazolam on sleep and bioamine metabolites in depression.

Alprazolam administered for 43 days in doses of 6 to 10 mg/day had an antidepressant effect in four of nine depressed patients. Decreases in slow wave sleep, increases in rapid eye movement (REM) latency, and decreases in REM minutes and percent and REM sleep eye movements were found in the group as a whole. The drug had a general hypnotic effect with a trend toward increased total sleep time. Nonsignificant changes in the concentrations of 3-methyl-4-hydroxyphenylglycol and homovanillic acid in the cerebrospinal fluid (CSF) were qualitatively similar to those found after treatment with tricyclic antidepressant drugs; however, only the larger decreases in CSF 5-hydroxyindoleacetic acid achieved statistical significance. Baseline sleep and CSF metabolites and changes in these measures on drug did not predict the therapeutic effects of alprazolam.

Adult↗
PubMed1993

Porcine cerebroside sulfate activator: further structural characterization and disulfide identification.

Cerebroside sulfate activator (CS-Act) is a small compact protein which binds and solubilizes certain glycosphingolipids. Following the recent publication of the purification and preliminary sequence of pig kidney CS-Act [Fluharty, A.L., Katona, Z., Meek, W.E., Frei, K., & Fowler, A.V. (1992) Biochem. Med. Metab. Biol. 47, 66-85], we now report the primary sequence of the C-terminal portion of this protein and the assignment of the three disulfide bonds. Cyanogen bromide (CNBr) treatment of native CS-Act produced three major and several minor peptide fragments. Analysis of one HPLC-purified fragment revealed the C-terminus 14 amino acid sequence. This established the length of the native protein at 79 residues. In conjunction with the sequence data for one other major HPLC-purified CNBr fragment, it could be concluded that the three intrachain disulfide bonds were located at half-cystine residues 4 and 77, 7 and 71, and 36 and 47. Mass spectrometry (fast atom bombardment and electrospray ionization) showed the molecular weight of the major component of the CS-Act preparation to be 9720.5 Da, which was in close agreement with the calculated mass of the 79 amino acid peptide with five covalently attached sugar residues and three internal disulfide bonds. The mass spectrometric molecular weight measurements also showed that the CS-Act preparation possessed microheterogeneity in its carbohydrate moiety, as less intense signals corresponded to species containing (in decreasing order of abundance) two, one, four, and three sugar residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Pharmacologic effects in vivo in brain by vector-mediated peptide drug delivery.

Pharmacologic effects in brain caused by systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium, which comprises the blood-brain barrier in vivo. In the present study, successful application of a chimeric peptide approach to enhance drug delivery through the blood-brain barrier for the purpose of achieving a central nervous system pharmacologic effect is described. The chimeric peptide was formed by linkage of a potent vasoactive intestinal peptide (VIP) analogue, which had been monobiotinylated, to a drug transport vector. The vector consisted of a covalent conjugate of avidin and the OX26 monoclonal antibody to the transferrin receptor. Owing to the high concentration of transferrin receptors on brain capillary endothelia, OX26 targets brain and undergoes receptor-mediated transcytosis through the blood-brain barrier. Systemic infusion of low doses (12 micrograms/kg) of the VIP chimeric peptide in rats resulted in an in vivo central nervous system pharmacologic effect: a 65% increase in cerebral blood flow. Biotinylated VIP analogue without the brain transport vector was ineffective.

Amino Acid Sequence↗