Biomedical subjects

A Kurosky

Publications and source records attributed to A Kurosky.

At least 37 records · Page 2Linked to original sources

PubMed1995

Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.

Amino Acid Sequence↗

PubMed1995

Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts.

A significant amount of host cellular annexin II was found to be associated with human cytomegalovirus isolated from cultured human fibroblasts (approximately 1,160 molecules per virion). This composition was established by four different analytical approaches that included (i) Western blot (immunoblot) analysis of gradient-purified virions with a monoclonal antibody specific for annexin II, (ii) peptide mapping and sequence analysis of virus-associated proteins and proteins dissociated from virus following EDTA treatment, (iii) electron microscopic immunocytochemistry of gradient-purified virions, and (iv) labeling of virus-associated proteins by lactoperoxidase-catalyzed radioiodination. These results indicated that annexin II was primarily localized to the viral surface, where it bound in a divalent cation-dependent manner. In functional experiments, a rabbit antiserum raised against annexin II inhibited cytomegalovirus plaque formation in human foreskin fibroblast monolayers in a concentration-dependent manner. Cumulatively, these studies demonstrate an association of host annexin II with cytomegalovirus particles and provide evidence for the involvement of this cellular protein in virus infectivity.

Animals↗

PubMed1994

Structural characterization of a Lymnaea putative endoprotease related to human furin.

A number of peptides have been identified in the central nervous system of the freshwater snail, Lymnaea stagnalis, that function as hormones and neurotransmitters/neuromodulators. These peptides are typically proteolytically processed from larger prohormones mostly at sites composed of single or multiple basic amino acid residues. Previously we demonstrated a diversity of putative prohormone convertases that may be involved in prohormone processing in the Lymnaea brain. In the present report, we have characterized a cDNA clone encoding a putative endoprotease of 837 amino acids. The primary structure of endoprotease (Lfur2) was comparable to that of human furin and contained a putative catalytic domain, a Cys-rich domain, and a transmembrane region. The catalytic domain of Lfur2 demonstrated about 70% residue identity when compared with human furin, PACE4 and Drosophila Dfur1 and dKLIP-1. The Lfur2 gene was expressed in the central nervous system as well as various peripheral tissues of Lymnaea.

Amino Acid Sequence↗

PubMed1994

An endothelial cell-surface form of annexin II binds human cytomegalovirus.

Human cytomegalovirus was shown to bind to human umbilical vein endothelial cells in a specific, saturable and calcium-dependent manner (Kd = 7.9 pM (4 degrees C), 6469 virus binding sites/cell). Affinity adsorption of detergent-prepared lysates of surface-radiolabeled endothelial cells to virions resulted in the identification of cell-derived proteins of approximate M(r) 36,000 and 32,000 that bound cytomegalovirus. Protein sequencing of peptides obtained by cyanogen bromide cleavage demonstrated that the 36 kDa protein corresponded to human annexin II, and the 32 kDa protein was likely a degradation product. Purified annexin II was demonstrated to bind directly to virions (Kd = 57 nM, 688 annexin II binding sites/virion). These results provide evidence that an endothelial cell-surface form of annexin II acts as a receptor for cytomegalovirus, and indicate a previously undescribed role for annexin II.

Amino Acid Sequence↗

PubMed1994

Identification and characterization of O-biotinylated hydroxy amino acid residues in peptides.

Recent findings have indicated that certain hydroxyl groups of peptides have enhanced intrinsic chemical reactivity and can be O-acylated by N-hydroxysuccinimide esters of biotin. Analytical procedures are described for the identification of O-acylated derivatives of seryl, threonyl, and tyrosyl residues after reaction with the N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid. The identification and chemical characterization of O-biotinylated residues included amino acid analysis, peptide sequence determination, and mass spectrometric analysis. The application of these analytical procedures provided unequivocal identification of O-biotinylated residues for a number of peptides investigated. In a separate study, we also show that O-biotinylated amino-terminal seryl residues occurring after proteolysis of peptides or proteins can rapidly undergo an O-->N acyl shift resulting in blockage of sequence analysis by the Edman degradation procedure.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of Aplysia atrial gland secretory granules containing egg-laying prohormone-related peptides.

A purification scheme is described for the isolation of secretory granules containing egg-laying prohormone-related peptides from the atrial gland of Aplysia californica, an exocrine organ in the reproductive tract. Granules were purified by differential centrifugation of atrial gland homogenates followed by centrifugation on continuous Percoll-sucrose gradients. Quantitative enzyme assays in conjunction with electron microscopic analyses demonstrated that secretory granules thus isolated were significantly purified with respect to other subcellular organelles such as mitochondria and lysosomes. Immunoelectron microscopy demonstrated that the majority (approximately 85%) of the purified secretory granules were immunoreactive for A-NTP (N-terminal peptide), a cleavage product of the egg-laying prohormone-related A and A' precursors (residues 22-34). The purified granules represented an enriched source of peptides that were readily resolved by reversed-phase high performance liquid chromatography.

Animals↗

PubMed1994

A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes.

We produced large quantities of the extracellular domain of the human TSH receptor (ETSHR) using the baculovirus expression system. Insect cells containing the ETSHR protein were sequentially extracted using lysis, nuclease, and high salt buffers to enrich for recombinant protein. The ETSHR protein was purified to homogeneity on a C4 reverse phase semipreparative column using HPLC. The recombinant protein was identified as ETSHR by immunoreactivity with antibodies prepared against TSHR-derived synthetic peptides. The identity of the ETSHR was further confirmed by amino acid compositional analyses, which agreed with the amino acid composition predicted from reported cDNA sequence analyses. Protein sequence analyses confirmed that the first 26 amino acids of the N-terminal region and the C-terminal amino acid were identical to the predicted amino acid sequence. The purified ETSHR was refolded in the presence of 1.5 M guanidine-HCl and 1 mM each of cystine and cysteine. [125I] TSH bound to the refolded ETSHR in vitro in a dose-dependent manner and was specifically blocked by unlabeled TSH, but not by LH or FSH. It was notable that a membrane requirement was not essential for TSH to bind to ETSHR.

Amino Acid Sequence↗

PubMed1993

Elevated intrinsic reactivity of seryl hydroxyl groups within the linear peptide triads His-Xaa-Ser or Ser-Xaa-His.

Chemical modification studies of peptide hormones and random peptides have revealed that seryl hydroxyl groups had enhanced reactivity toward acylating reagents when they occurred in the linear triads His-Xaa-Ser or Ser-Xaa-His (Xaa = any amino acid). O-acylation of serine within these triads was achieved by reaction with N-hydroxysuccinimide esters of biotin (NHS-biotin) and succinic anhydride. Seryl residues not occurring in His-Xaa-Ser/Ser-Xaa-His triads showed no reactivity towards NHS-biotin under reaction conditions described. Results of histidine replacement studies and studies of the pH dependence of O-biotinylation indicated that the increased nucleophilicity of the seryl hydroxyl group was due to intramolecular interaction between the seryl and histidyl residues. Our findings provide strong evidence that such triads represent novel consensus motifs in peptides.

Amino Acid Sequence↗

PubMed1993

A conserved binding site on the receptor for polymeric Ig is homologous to CDR1 of Ig V kappa domains.

We have previously shown that the binding of human secretory component (SC) to polymeric IgA (PIgA) is inhibited by a synthetic peptide fragment of SC (SC[15-37]), and also by a mAb (mAb 6G11) which specifically recognizes the unbound form of SC and SC[15-37]. In this report we show that the binding of bovine, rabbit, and rat SC to human PIgA can also be inhibited by SC[15-37], and that the relative affinity of these three species of SC for human PIgA correlated with their relative affinity for mAb 6G11. The binding of mAb 6G11 to SC isolated from various species was especially affected by amino acid substitutions at position 26 of SC. Structural modeling revealed that the side chain of residue 26 is located on the external face of a loop in SC domain I which is homologous in size and position to the first complementarity determining region (CDR1) of Ig V kappa domains. However, the surface topography of the CDR1-like loop of SC is distinct from other known V kappa CDR1 loops due to the unique distribution of charged and bulky polar side chains which locate to the exterior face of the loop. Together, these findings indicated that the binding site on the receptor for PIg is highly conserved among species, and is composed, in part, of residues of domain I of SC which may form a distinct CDR1-like loop on the surface of the molecule.

Amino Acid Sequence↗

PubMed1993

Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.

A procedure employing C18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method, we determined the observed pseudo-first-order reaction rate constant (k'1) of biotinylation of lysyl residues in two model peptides, [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22.10(-2)s-1 and 1.08.10(-2)s-1, respectively, in 0.05 M sodium phosphate buffer, pH 8.2, at 25 degrees C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic and alkaline pH and for evaluating biotin reagent homogeneity.

Amino Acid Sequence↗

PubMed1993

Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells.

1. Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases. 2. Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/Drosophila furin, human PC2, and mouse PC1/PC3 and PC2. 3. The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%). 4. These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland. 5. Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes.

Amino Acid Sequence↗

PubMed1992

The occurrence of O-acylation during biotinylation of gonadotropin-releasing hormone and analogs. Evidence for a reactive serine.

Gonadotropin-releasing hormone (GnRH) and two of its analogs ([D-Lys6]GnRH and des-Gly10-[D-Trp6]-GnRH) were reacted with sulfonated N-hydroxysuccinimide esters of biotin that have been reported to react specifically with primary amino groups. Fractionation by reversed-phase high performance liquid chromatography demonstrated the occurrence of multiple biotinylated derivatives for each reacted peptide. These results were unexpected since GnRH and des-Gly10-[D-Trp6]GnRH contained no reactive amino groups and [D-Lys6]GnRH had only one. Reaction of the biotinylated derivatives with hydroxylamine indicated that significant O-biotinylation had occurred. Mass spectrometric analyses established the stoichiometry of biotinylation and confirmed that substantial O-biotinylation of residue Ser4, and to a minor extent Tyr5, of GnRH and the two analogs had occurred. In contrast, the biotinylation of selected peptides unrelated to GnRH under identical reaction conditions indicated no significant evidence of O-acylation of seryl residues. Strikingly, biotinylation of GnRH under denaturing conditions largely abolished O-acylation, indicating that the observed O-biotinylation was dependent on peptide conformation. All the O-biotinylated derivatives displayed significantly reduced bioactivity. Taken together, these results give strong evidence that the Ser4 hydroxyl of GnRH has a significantly elevated intrinsic reactivity, which raises new questions concerning its putative role in the conformation and mode of action of the hormone. These results also demonstrate for the first time that the N-hydroxysuccinimide-biotin esters are capable of significant O-acylation and may be generally useful reagents for detecting highly reactive hydroxyamino acid residues.

Acylation↗

PubMed1991

N-acylation of Aplysia egg-laying hormone with biotin. Characterization of bioactive and inactive derivatives.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

Acylation↗

PubMed1991

Characterization of a critical binding site for human polymeric Ig on secretory component.

Secretory component (SC) is an integral membrane glycoprotein of secretory epithelial cells which is responsible for the specific transport of polymeric Ig (PIg) to external mucosal surfaces. The ectoplasmic segment which binds polymeric Ig is comprised of five Ig-type domains. Chemically and enzymatically modified forms of the ectoplasmic portion of SC (FSC) were produced and tested for their ability to bind to PIgA and PIgM. Deglycosylated FSC bound specifically to PIg, indicating that N-linked carbohydrate moieties on FSC are not required for binding. Denatured, reduced, and alkylated FSC did not bind to PIgA, and bound to PIgM with significantly reduced affinity, suggesting that native conformation of the polypeptide backbone of SC was important to binding. Tryptic fragments of FSC which bound to PIg were isolated and identified to be derived from domain I of SC. Synthetic peptides comprising overlapping portions of domain I bound to PIg to varying degrees. The strongest affinity was demonstrated by a peptide comprised of residues 15 to 37 of SC. A comparison of the amino acid sequences of human, rabbit, and rat SC indicated that this region contained a high degree of residue identity (78%) and may represent a consensus sequence for binding of FSC to PIg. Importantly, the peptide comprised of residues 15 to 37 was also recognized by a monoclonal antibody, 6G11, which inhibited the binding of FSC to PIgA. These results demonstrate that the binding of human SC to PIg is critically dependent on a highly conserved peptide region within the first domain of SC centering at residues 15-37.

Amino Acid Sequence↗

PubMed1991

Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization.

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

Amino Acids↗

PubMed1991

Biotinylated peptides/proteins. II. Identification of biotinylated lysyl phenylthiohydantoins.

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.

Amino Acid Sequence↗

PubMed1991

Review of Aeromonas enterotoxins.

This report reviews the work of other investigators regarding Aeromonas toxins and describes work conducted in our laboratory relating to the biochemical characterization of a cytolytic factor with an antigenic moiety that cross-reacts with cholera toxin (referred to as CTC-cytolysin), as well as the purification and partial characterization of a non-CTC enterotoxin. These two toxins were produced by Aeromonas hydrophila, isolate SSU, and are capable of causing fluid accumulation in animal models.

Aeromonas↗

PubMed1991

Genetic variation in related cytolytic toxins produced by different species of Aeromonas.

Comparative analyses were performed at the immunological, biological, and genetic level to establish the degree of relatedness of a group of four cytotoxic enterotoxins and aerolysins produced by different isolates of Aeromonas: SSU [16], Ah1 [13], Ah2 [18], and Ah65 [21]. Results obtained from Western blot analysis, neutralization studies, and Southern blot analysis indicated that although the members of this group of toxins displayed structural similarities they also differed from each other at the immunological, biological and genetic level.

Aeromonas↗