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F Naider

Publications and source records attributed to F Naider.

At least 91 records · Page 5Linked to original sources
PubMed1989

Biologically significant conformation of the Saccharomyces cerevisiae alpha-factor.

The conformation of the tridecapeptide alpha-factor of the yeast Saccharomyces cerevisiae was examined in both solution and in the presence of lipid vesicles. CD, differential scanning calorimetry, and phosphorus nmr all indicate that this mating pheromone interacts with lipid vesicles. In both aqueous and organic solution the alpha-factor is a flexible molecule that exhibits features of a type II beta-turn spanning the center of the peptide. Two-dimensional Nuclear Overhauser enhancement spectroscopy gives evidence that the beta-turn is stabilized on interaction of the peptide with lipid vesicles. Our current belief is that the beta-turn may play an important role in the biologically active conformation of the alpha-factor.

Mating Factor↗
PubMed1988

Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae.

alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.

Binding, Competitive↗
PubMed1988

Partial characterization and purification of the glycosylation site recognition component of oligosaccharyltransferase.

Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.

Affinity Labels↗
PubMed1987

Synthesis and biological activity of amino terminus extended analogues of the alpha mating factor of Saccharomyces cerevisiae.

The synthesis and biological activity are reported for extended analogues of the secreted tridecapeptide alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) from Saccharomyces cerevisiae. Peptides with Ala, Glu-Ala, Ala-Glu-Ala, or Glu-Ala-Glu-Ala attached to the amino terminus of alpha-factor were synthesized by the solid-phase method on a (phenylacetamido)methyl (PAM) resin, using a combination of dicyclohexylcarbodiimide- and 1-hydroxybenzotriazole-accelerated active ester coupling procedures. Free peptides were obtained by hydrogen fluoride (HF) cleavage in the presence of appropriate scavengers. Normal high HF cleavage and "low-high" HF cleavage were equally effective in liberating the desired product from the PAM resin. Yields of pure peptide ranged from 9% to 17%. All of the extended alpha-factors, which represent sequences of pro-alpha-factor coded for in the MF alpha 1 structural gene, caused morphological aberrations (shmoo assay) in strain X2180-1A (MATa) the same as those caused by the tridecapeptide. The 14-peptide was equally active compared to the native alpha-factor whereas the 17-peptide was 5-10-fold less active. The analogues also arrested to various degrees (halo assay) the growth of S. cerevisiae RC629 (MATa sst1) and S. cerevisiae RC631 (MATa sst2), two supersensitive mutants, and were converted to pheromones of equal activity by treatment with V8 protease. A temperature-sensitive receptor mutant responded to all the peptides at the permissive but not the restrictive temperature. An alpha-factor antagonist, des-Trp1,Ala3-alpha-factor, inhibited activity of all extended peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Chemochromatography: its use for the separation of nikkomycins X and Z.

A novel mode of reversed-phase high-performance liquid chromatography in which the mobile phase reacts chemically with the compounds to be separated was developed. Nikkomycin X and nikkomycin Z, two natural isomeric nucleoside peptide antibiotics, move as a single peak on a C18 reversed-phase column using an aqueous trifluoroacetic acid mobile phase. Addition of sodium bisulfite (1.0%) to the mobile phase results in the formation of a polar bisulfite addition product with nikkomycin X, but not with nikkomycin Z, inside the HPLC column. This type of reactive chromatography, or chemochromatography, led to the analytical and preparative separation of nikkomycins X and Z which are normally very intractable to separation by conventional chromatographic techniques.

Aminoglycosides↗
PubMed1987

Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations.

Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides. We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 microM (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-[3H]leucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures. We concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids.

Amino Acids↗
PubMed1987

Towards determination of the structure of the Saccharomyces cerevisiae a-factor: an acylated pentadecapeptide blocks a-factor activity.

Putative a-factor peptides YIIKGVFWADP, YIIKGVFWANP, YIIKGLFWADP, YIIKGLFWANP, YIIKGVFWDPA, and YIIKGVFWDPACVIA and several peptide derivatives were synthesized and were found to be inactive in growth arrest assays, yet they blocked the activity of biological a-factor. Antagonism was greatest with YIIKGVFWDPAC(palmitoyl)VIA. Thus, the structure of a-factor may be a lipopeptide resembling this palmitoylated pentadecapeptide.

Acylation↗
PubMed1986

Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe.

Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.

Affinity Labels↗
PubMed1986

Yeast alpha-factor and somatostatin enhance binding of [3H]estradiol to proteins in rat pancreas and Saccharomyces cerevisiae.

Pancreatic tissue contains an [3H]estradiol-binding protein that requires a coligand in the steroid-binding reaction. The endogenous coligand appears to be the tetradecapeptide somatostatin. Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of [3H]estradiol to partially purified pancreatic protein. Supernatant fractions from yeast cells also contain an [3H]estradiol-binding protein. alpha-Factor can enhance specific binding of [3H]estradiol to such yeast fractions. Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of [3H]estradiol but did not inhibit cell growth or induce morphological changes in S. cerevisiae. Thus, it appears that coligand-requiring [3H]estradiol-binding activity and mating in yeast are not directly related.

Amino Acid Sequence↗
PubMed1986

Synthesis and biological properties of chitin synthetase inhibitors resistant to cellular peptidases.

The synthesis and biological properties of seven polyoxins (4-10) designed to avoid peptidase hydrolysis in Candida albicans are presented. Five dipeptidyl and two tripeptidyl polyoxin analogues were synthesized by coupling an amino acid active ester or azlactone to uracil polyoxin C (2) or polyoxin D (1), subsequent removal of the protecting group, and purification by preparative HPLC. A new and novel route for introducing an n-propyl group onto the alpha-amino group of peptides is reported. With the exception of a carboxamide derivative, 8, all analogues were resistant to hydrolysis by a cell extract or permeabilized cells of Candida. Chitin synthetase inhibition constants were determined for 4-10 and the KI values ranged from 7.15 X 10(-6) M for octanoyl-phenylalanyl-polyoxin D (10) to 1.06 X 10(-3) M for D-tryptophanyl-uracil polyoxin C (6). These novel polyoxins do not compete with the transport of either peptides or uridine into the cell. Millimolar concentrations of compounds 4-10 are required to inhibit growth, cause morphological alterations, or reduce the viability of C. albicans.

Antifungal Agents↗
PubMed1986

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans.

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.

Antifungal Agents↗
PubMed1986

Hydrophobic polyoxins are resistant to intracellular degradation in Candida albicans.

Two novel polyoxins, N-epsilon-(octanoyl)-lysyl-uracil polyoxin C (Oct-Lys-UPOC) and N-gamma-(octyl)-glutaminyluracil polyoxin C (Oct-Gln-UPOC), were synthesized by reacting uracil polyoxin C with the appropriate amino acid p-nitrophenyl ester. Oct-Lys-UPOC and Oct-Gln-UPOC were strong inhibitors (Kis = 1.7 X 10(-6)M) of chitin synthetase from Candida albicans membrane preparations. In a permeabilized-cell assay, Oct-Gln-UPOC had a 10-fold-lower inhibitory activity toward chitin synthetase than did the Oct-Lys-UPOC analog. Both compounds were resistant to hydrolysis by a cell extract of C. albicans H317; however, Oct-Gln-UPOC was hydrolyzed with a half-life of 23 min by a permeabilized-cell preparation. Oct-Lys-UPOC was resistant to hydrolysis by permeabilized cells. Oct-Gln-UPOC and Oct-Lys-UPOC did not compete with the transport of peptides or uridine into the cell. At concentrations up to 2 mM these two new polyoxins were ineffective in the inhibition of cell growth or reduction of cell viability, but they induced aberrant morphologies in C. albicans at a concentration of 0.25 mM. These data suggest that polyoxins containing hydrophobic amino acids retain strong chitin synthetase inhibitory activity and are resistant to cellular hydrolysis. They provide the first example of effective synthetic chitin synthetase inhibitors which are stable inside C. albicans.

Anti-Bacterial Agents↗
PubMed1986

Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae.

The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH). To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis. Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide. The activities of the two peptides were additive and indistinguishable for S. cerevisiae X2180-1A. When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides. Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature. These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.

Amino Acid Sequence↗
PubMed1986

Structure-activity relationships of the yeast alpha-factor.

The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation. Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied. These peptides have been analyzed for their biological activities by various assays. Mutants of S. cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues. The mating system of S. cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.

Amino Acid Sequence↗
PubMed1985

Biological activity and conformational isomerism in position 9 analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor from Saccharomyces cerevisiae.

Analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor of Saccharomyces cerevisiae, where the glycyl residue of position 9 was replaced by D-Ala, L-Ala, D-Leu, and L-Leu, were synthesized and evaluated by morphogenesis assays and circular dichroism spectroscopy. Synthesis was accomplished in solution phase with mixed anhydrides and p-nitrophenyl active esters as the coupling agents. All crude dodecapeptides were purified to greater than 98% homogeneity by preparative high-performance liquid chromatography on a reversed-phase column. The Gly9, D-Ala9, and D-Leu9 analogues elicited morphogenic alterations in MATa strains of S. cerevisiae at concentrations of 1-2 micrograms/mL and exhibited similar CD patterns in both trifluoroethanol and tris(hydroxymethyl)aminomethane buffer, pH 7.4. In contrast, the L-Ala9 and L-Leu9 analogues were more than 200 times less active in the morphogenesis assay and had markedly different CD spectra. These results demonstrate that the position 9 residue plays an important role in determining the biological activity and solution conformation of alpha-factor. We suggest the presence of a type II beta-turn in the Lys7-Gln10 region when the alpha-factor assumes its biologically active conformation.

Alanine↗
PubMed1985

Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability.

Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents. Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase. Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells. These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity. All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability. These results and other structure-activity relationships [Baffi, R. A., Shenbagamurthi, P., Terrance, K., Becker, J. M., Naider, F., & Lipke, P. (1984) J. Bacteriol. 158, 1152-1156] indicate that the agglutination and morphological responses to alpha-factor can be varied independently. Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity. Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation. All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.

Agglutination↗