Biomedical subjects

W R Abrams

Publications and source records attributed to W R Abrams.

At least 37 records · Page 2Linked to original sources

PubMed1998

Submandibular salivary proteases: lack of a role in anti-HIV activity.

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.

Anti-HIV Agents↗

PubMed1998

Molecular cloning and characterization of the bovine and human tuftelin genes.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization.

Alternative Splicing↗

PubMed1998

Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis.

Saliva is an enriched milieu containing biologically active proteins, including several different growth factors and cytokines. This study documents that vascular endothelial growth factor (VEGF), a potent, multifunctional, angiogenic cytokine, is a component of normal human saliva. VEGF was measured by ELISA in whole saliva (median concentration, 460 pg/ml) and in ductal secretions obtained from the parotid (277 pg/ml) and the submandibular-sublingual (80 pg/ml) salivary glands. VEGF seems to be synthesized endogenously by the salivary glands because both VEGF mRNA and protein (as revealed by in situ reverse transcriptase-PCR and by immunohistochemistry, respectively) colocalized to serous acinar cells and ductal epithelial cells within the parotid, submandibular, and minor salivary glands. These findings point to the existence of a "salivary VEGF system." It is possible that salivary VEGF plays a role in regulating physiologic and pathologic angiogenic and other vascular responses in salivary and mucosal tissues. And in particular, the presence of VEGF in saliva may contribute to the remarkable healing capacity of the oral mucosa as well as other regions of the digestive tract.

Adult↗

PubMed1998

Molecular analysis of collagens in bladder fibrosis.

PURPOSE: Fibrosis of bladder tissue is characterized by an abnormal deposition of connective tissue within different layers of the bladder wall, resulting in a low volume, high pressure vesical which may ultimately contribute to renal scarring and failure. These bladders are functionally referred to as "non-compliant" and may result from different etiologies: neurogenic, which encompasses myelodysplasia and spinal cord injury, or non-neurogenic, owing to obstruction or radiation therapy. To examine the molecular mechanisms responsible for this fibrosis, we have analyzed a well-characterized pediatric patient population for alteration(s) in collagen types I and III regulation at the protein and nucleic acid levels. MATERIALS AND METHODS: Immunohistochemical localization of collagen subtypes (I, III, and IV) was carried out using type specific monoclonal antibodies. Total collagen was determined by hydroxyproline analysis, and subtype specific expression of collagenous proteins, following cyanogen bromide extraction procedures, was quantified by competitive ELISA. Total RNA was extracted by guanidinium/phenol/chloroform, and slot blot hybridization analyses with radiolabeled human cDNA probes were quantified by densitometry of resulting autoradiograms. RESULTS: Connective tissue infiltration of detrusor smooth muscle bundles was specific for type III collagen. Protein analyses demonstrated: 1) an increase in total collagen, 2) a statistically significant increase in the type III: type I collagen ratio, and 3), an absolute increase in type III collagen protein in non-compliant bladder tissue. At the mRNA level, there was a coordinate increase in both collagen I and III steady-state mRNAs in non-neurogenic bladder tissue, whereas neurogenic bladder tissue was characterized by an increase in the type III: type I mRNA transcript ratio. CONCLUSIONS: These data suggest that regulation of collagen synthesis in bladder fibrosis is complex and is characterized by both transcriptional and post-transcriptional mechanisms, depending upon the etiology of the fibrosis.

Adolescent↗

PubMed1997

Inhibitors and specificity of Pseudomonas aeruginosa LasA.

LasA is an extracellular protease of Pseudomonas aeruginosa that enhances the elastolytic activity of Pseudomonas elastase and other proteases by cleaving elastin at unknown sites. LasA is also a staphylolytic protease, an enzyme that lyses Staphylococcus aureus cells by cleaving the peptidoglycan pentaglycine interpeptides. Here we showed that the staphylolytic activity of LasA is inhibited by tetraethylenepentamine and 1,10-phenanthroline (zinc chelators) as well as excess Zn2+ and dithiothreitol. However, LasA was not inhibited by several serine or cysteine proteinase inhibitors including diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin, and N-ethylmaleimide. LasA staphylolytic activity was also insensitive to Nalpha-p-tosyl-L-lysine chloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory only at concentrations greater than 20 mM. Without added inhibitors, LasA obtained by DEAE-cellulose fractionation was active toward beta-casein, but the same cleavage patterns were observed with column fractions containing little or no LasA. The beta-casein cleaving activity was fully blocked in the presence of inhibitors that did not affect staphylolytic activity. In the presence of such inhibitors, purified LasA was inactive toward acetyl-Ala4 and benzyloxycarbonyl-Gly-Pro-Gly-Gly-Pro-Ala, but it degraded soluble recombinant human elastin as well as insoluble elastin. N-terminal amino acid sequencing of two fragments derived from soluble elastin indicated that both resulted from cleavages of Gly-Ala peptide bonds located within similar sequences, Pro-Gly-Val-Gly-Gly-Ala-Xaa (where Xaa is Phe or Gly). In addition, Ala was identified as the predominant N-terminal residue in fragments released by LasA from insoluble elastin. A dose-dependence study of elastase stimulation by LasA indicated that a high molar ratio of LasA to elastase was required for significant enhancement of elastolysis. The present results suggest that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin. Substrates that contain no Gly-Gly peptide bonds such as beta-casein appear to be resistant to LasA.

Bacterial Proteins↗

PubMed1997

Molecular cloning and characterization of the bovine tuftelin gene.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to that of bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014-bp open reading frame encoding a protein of 338 residues with a calculated mol. wt of 38,630 and an isoelectric point of 5.85. These results differ from those previously published, (1991) which contained a different conceptual amino acid sequence for the carboxy terminal region and identified a different termination codon. The protein does not appear to share homology or domain motifs with any other known protein. The gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxyterminal and 3' untranslated regions. The exons are embedded in more than 28 kbp of genomic DNA. Codons are generally not divided at exon/intron borders. Several alternatively spliced transcripts were identified by DNA sequence analysis of the isolated products produced by reverse transcriptase/polymerase chain reaction.

Alternative Splicing↗

PubMed1997

Expression of microfibrillar proteins by bovine bladder urothelium.

OBJECTIVES: To determine the occurrence and potential function of proteins composing elastic microfibrils in the developing bovine bladder. METHODS: Monospecific antibodies, generated against two well-characterized microfibrillar proteins, microfibril-associated glycoprotein (MAGP) and fibrillin-1 (FBN1), were used in immunohistochemical analysis of full-thickness frozen sections of fetal bovine bladder. The localization of these two antibodies was compared with that of anti-type IV collagen antibody. Adjacent serial sections were stained for routine light microscopy. Cultured urothelial cells were fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 before immunoanalysis. Control reactions used either preimmune serum or a monoclonal antibody to a nonmatrix protein. Poly(A+) ribonucleic acid was isolated from cultured urothelial cells and subjected to Northern analysis using specific complementary deoxyribonucleic acid probes for MAGP and FBN1. RESULTS: Both MAGP and FBN1 are expressed by the urothelium and are found in association with the underlying basement membrane, as visualized by their co-localization with type IV collagen. Furthermore, urothelial cells in culture continue to express both microfibrillar proteins. CONCLUSIONS: The developing bovine urothelium expresses major microfibrillar protein components. The role of these microfibrils in the urothelium remains to be determined, but they may have an important anchoring function.

Animals↗

PubMed1997

Inhibition of HIV infectivity by human saliva.

OBJECTIVE: Human saliva is known to decrease HIV infectivity in vitro. The purpose of this study was to extend these findings and to focus on the mechanism of action of these salivary factor(s). DESIGN: A number of viruses and several assay systems have been utilized to determine if the effect of submandibular saliva is directly on the virus, on the host cell, or on the virus-cell interaction. MATERIALS AND METHODS: Submandibular saliva from seronegative donors was incubated with HIV-1, other retroviruses, or unrelated viruses. Viral infectivity was monitored either by determining p24 antigen levels in peripheral blood mononuclear cells or Sup T1 cells, or using HeLa cells expressing CD4 and an HIV derived long terminal repeat linked to the beta-galactosidase gene. RESULTS: The inhibition of viral infectivity by submandibular saliva is specific for HIV-1. While inhibition increases with time of incubation of saliva with virus, pretreatment of cells with saliva does not inhibit HIV production, and saliva has only modest inhibitory effects when added to HIV-infected cells. CONCLUSIONS: It appears that the effect of submandibular saliva on decreasing the infectivity of HIV-1 is directly on the virus, rather than on the host cell.

Antiviral Agents↗

PubMed1997

Stabilization of elastin mRNA by TGF-beta: initial characterization of signaling pathway.

The cytokine transforming growth factor-beta (TGF-beta) has multiple effects on a wide variety of cell types. These effects include modulation of growth and regulation of gene transcription. In a few instances, TGF-beta has also been shown to regulate gene expression posttranscriptionally by altering message stability, but the pathway by which this activity is executed remains largely unknown. In the present work, we demonstrate that TGF-beta 1 has no effect on transcription of the elastin gene in cultured human fetal lung fibroblasts, but does stabilize elastin messenger RNA (mRNA), leading to a dramatic increase in the steady-state level of elastin mRNA. A corresponding increase in production of tropoelastin accompanies the increase in elastin mRNA. Through the use of specific inhibitors, we demonstrate that phosphatidylcholine (PC)-specific phospholipase C (PLC) and protein kinase C (PKC) are involved in mediating the elastin message stabilization. In contrast, G proteins and extracellularly regulated kinases do not appear to be involved. These results suggest that although the TGF-beta signaling pathway leading to message stabilization shares components with that modulating transcription, the message-stabilization pathway also contains diverse other elements.

Bridged-Ring Compounds↗

PubMed1996

Molecular cloning and expression of the gene for elastin-binding protein (ebpS) in Staphylococcus aureus.

Interactions between staphylococci and components of the extracellular matrix mediate attachment of the bacteria to host tissues and organs and define an important mechanism leading to colonization, invasion, and formation of metastatic abscesses. We have previously demonstrated a specific binding interaction between Staphylococcus aureus and elastin, one of the major protein components of the extracellular matrix. Available evidence suggests that this association is mediated by a 25-kDa elastin-binding protein on the surface of S. aureus (EbpS). To study the molecular structure and function of EbpS, the gene encoding EbpS was cloned, sequenced, and expressed in Escherichia coli. DNA sequence data indicate that the ebpS open reading frame consists of 606 base pairs and encodes a novel polypeptide with a predicted molecular mass of 23,345 daltons and pI of 4.9. A polyclonal antibody raised against recombinant EbpS interacted with the native 25-kDa cell surface EbpS and inhibited staphylococcal elastin binding. Furthermore, recombinant EbpS bound specifically to immobilized elastin and inhibited binding of S. aureus to elastin. A degradation product of recombinant EbpS lacking the first 59 amino acids of the molecule and a C-terminal fragment of CNBr-cleaved recombinant EbpS, however, did not interact with elastin. Together, these results confirm that EbpS is the cell surface molecule mediating binding of S. aureus to elastin. The inability of truncated forms of recombinant EbpS to bind to elastin suggests that the elastin binding site in EbpS is contained in the first 59 amino acids of the molecule.

Amino Acid Sequence↗

PubMed1996

Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags.

Expression of recombinant eukaryotic proteins in transfected mammalian cell lines has become an important approach for the characterization of the structure and function of these proteins. However, it is often difficult to recover and purify the recombinant proteins. Therefore, the use of fusion proteins incorporating epitope or affinity tags has become more widespread. In this paper, we directly compare two affinity tags, the hexahistidyl tag and the biotin peptide mimetic, Strep-tag, for use in purification of a recombinant soluble form of rabies virus glycoprotein secreted by transfected Chinese hamster ovary fibroblasts. The recombinant rabies virus glycoproteins are denoted RGP(WT)T441his and RGP(WT)T443s-tag, respectively. These affinity tags were chosen because the chromatographic matrices (Ni(II)-NTA-agarose and recombinant core streptavidin-agarose, respectively) were readily available and these methods offered the possibility of a one-step purification using mild elution conditions. However, in our hands, neither method allowed for a one-step purification protocol. Nonetheless, it was possible to purify RGP(WT)T441his to homogeneity from crude conditioned medium using a combination of metal-chelate affinity chromatography and immunoaffinity chromatography. In contrast, although the Strep-tag has been useful for purifying recombinant proteins expressed in bacteria, we were not able to effectively purify RGP(WT)T443s-tag from conditioned medium using chromatography on recombinant core streptavidin-agarose.

Affinity Labels↗

PubMed1996

Analysis of the human gene encoding latent transforming growth factor-beta-binding protein-2.

Transforming growth factor (TGF)-beta is secreted as an inactive complex, which frequently contains a large molecular weight binding protein designated latent TGF-beta-binding protein (LTBP). Recently, the LTBPs have been shown to be a gene family that contains three known members and exhibits a multidomain structure containing cysteine-rich motifs that are also found in the fibrillin gene family. The present work seeks to characterize the gene encoding LTBP-2 and to compare its features to that of the other LTBPs and to the fibrillins. Human fibroblast libraries were used to isolate cDNA encoding LTBP-2 which was then used to identify LTBP-2 transcripts and to isolate the corresponding LTBP-2 gene. The cloned cDNA encodes a 195 kDa protein containing 20 epidermal growth factor (EGF)-like repeats, three repeats containing eight cysteines, and one segment that appears to be a hybrid of the two. Single exons encode EGF repeats while the eight-cysteine repeats are encoded in two exons. Northern analysis identified two transcripts of 7.5 and 9.0 kb, with the presently analyzed cDNA probably corresponding to the 7.5 transcript. Phylogenetic sequence comparisons demonstrated that LTBP-3 is more similar to LTBP-1 than LTBP-2, while LTBP-2 shows the most similarity to the fibrillins. These analyses suggest that LTBP-1 diverged from LTBP-3, and that LTBP-2 diverged from LTBP-1. Within the fibrillin family, fibrillin-1 is nearest to the LTBPs. While the domain structure of LTBP-2 is similar to that of the other LTBPs, LTBP-2 possesses unique regions that make it the largest member of the LTBP family. LTBP-2 may have dual functions as a member of the TGF-beta latent complex and as a structural component of microfibrils.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus.

A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of the microfibrillar protein MFAP3 and assignment of the gene to human chromosome 5q32-q33.2.

Microfibrils having a diameter of 10-12 nm, found either in association with elastin or independently, are an important component of the extracellular matrix of many tissues, but characterization of these microfibrils is incomplete. To further our understanding of the gene structure of proteins composing the microfibrils and to identify their chromosomal location, we have cloned and characterized another microfibril protein, designated microfibril-associated protein-3 (MFAP3). The human gene encoding MFAP3 has a very simple structure, containing only two translated exons encoding a protein of 362 amino acids. Monospecific antibodies prepared against the recombinantly expressed protein reacted with the microfibrils found in ocular zonules. MFAP3 does not appear to share homology with any other known protein. The gene was found to be located on chromosome 5q32-q33.2, near the locus 5q21-q31 reported for the fibrillin gene, FBN2, which has been linked to congenital contractural arachnodactyly. MFAP3 is a candidate gene for heritable diseases affecting microfibrils.

Amino Acid Sequence↗

PubMed1995

Structure of the elastin gene.

The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Large segments of the sequence are conserved but there are also considerable variations which range in extent from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and largescale deletions and insertions. In general, smaller differences are found among mammalian tropoelastins and greater ones between chicken and mammalian tropoelastins. Although only a single elastin gene is found per haploid genome, the primary transcript is subject to considerable alternative splicing, resulting in multiple tropoelastin isoforms. Functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the gene. The introns of the human gene are rich in Alu repetitive sequences, which may be the site of recombinational events, and there are also several dinucleotide repeats, which may exhibit polymorphism and, therefore, be effective genetic markers. The 5' flanking region is G+C rich and contains potential binding sites for numerous modulating factors, but no TATA box or functional CAAT box. The basic promoter is contained within a 136 bp segment and transcription is initiated at multiple sites. These findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.

Alternative Splicing↗

PubMed1995

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Adhesiveness↗

PubMed1994

Microfibril-associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase.

Microfibril-associated glycoprotein (MAGP) is an integral component of microfibrillar structures that play a critical role in the organization of elastic fibers in the extracellular matrix. To study possible molecular interactions between MAGP and other elastic fiber components, we have generated native MAGP using a baculovirus expression system and tested its ability to associate with tropoelastin and fibrillin. MAGP produced by SF9 cells underwent processing similar to the mammalian protein, including correct cleavage of the signal peptide and sulfation of tyrosine residues. When tested in solid-phase binding assays, native MAGP specifically bound to tropoelastin but not fibrillin-1. Binding to tropoelastin was divalent cation-independent and was completely blocked by reduction and alkylation of either protein. Antibody inhibition studies indicated that the carboxyl terminus of tropoelastin mediated its interaction with MAGP. In addition to binding to elastin, MAGP was also a substrate for transglutaminase, which might explain its propensity to form high molecular weight aggregates that cannot be dissociated with reduction or denaturation. Together, the results of this study provide new insights into the functional relationship between microfibrillar proteins and have important implications for understanding elastic fiber assembly.

Amino Acid Sequence↗

PubMed1994

5-Chloro[1,4-13C]levulinic acid modification of mammalian and bacterial porphobilinogen synthase suggests an active site containing two Zn(II).

5-Chloro[1,4-13C]levulinic acid ([1,4-13C]CLA) is an active site-directed inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrically condenses two molecules of 5-aminolevulinic acid (ALA) which are called A-side ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. [1,4-13C]CLA modifies bovine PBGS at the A-side ALA binding site. The C4 chemical shift indicates an intact keto moiety; the C1 chemical shift indicates a deprotonated carboxyl group. In contrast, [1,4-13C]CLA modification of Escherichia coli PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C1 chemical shifts indicate substantially deprotonated carboxylic acid groups. For one of four observed forms of [1,4-13C]CLA-modified E. coli PBGS, an analog of the P-site Schiff base is found. Bovine and E. coli PBGS contain two different zincs, ZnA and ZnB. Past results placed ZnA near A-side ALA. [1,4-13C]CLA modifies E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine cluster implicated in binding ZnB. This result places ZnB near P-side ALA. E. coli PBGS binds a third type of divalent metal, MgC or MnC, which is found to have no significant effect on the 13C NMR spectrum of the [1,4-13C]CLA-modified protein.

Amino Acid Sequence↗