Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies.

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C(H)3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose.

CAMPATH-1H is a recombinant humanized murine monoclonal immunoglobulin (IgG1) which recognizes the CDw52 antigen of human lymphocytes, and has been the subject of clinical trials for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. Peptide mapping by liquid chromatography-mass spectrometry was used to confirm the predicted amino acid sequences and profile glycosylation for two CAMPATH isotypes expressed in a murine myeloma cell line (NS0) and a single isotype expressed in both Chinese hamster ovary (CHO) and NS0 lines. The three major glycoforms identified in CAMPATH are fucosylated biantennary structures, containing zero, one, or two galactose residues. Glycosylation of the IgG1 form of CAMPATH expressed in CHO cells is consistent with normal human IgG. However, IgG1 and IgG4 expressed in NS0 cells include two potentially immunogenic glycoforms which contain either one or two nonreducing terminal alpha-linked galactose residues. Oligosaccharide structures were characterized by a combination of tandem mass spectrometry, methylation analysis, and exoglycosidase digestion. The strategy used here is designed to be widely applicable to recombinant glycoproteins.

Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif.

Three sites of N(G),N(G)-arginine methylation have been located at residues 205, 217, and 224 in the glycine-rich, COOH-terminal one-third of the HeLa A1 heterogeneous ribonucleoprotein. Together with the previously determined dimethylated arginine at position 193 [Williams et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670], it is evident that all four sites fall within a span of sequence between residues 190 and 233 that contains multiple Arg-Gly-(Gly) sequences interspersed with phenylalanine residues. These RGG boxes have been postulated to represent an RNA binding motif [Kiledjian and Dreyfuss (1992) EMBO J. 11, 2655-2664]. Dimethylation of HeLa A1 appears to be quantitative at each of the four positions. Arginines 205 and 224 have been methylated in vitro by a nuclear protein arginine methyltransferase using recombinant (unmethylated) A1 as substrate. This suggests A1 may be an in vivo substrate for this enzyme. Examination of sequences surrounding the sites of methylation in A1 along with a compilation from the literature of sites that have been identified in other nuclear RNA binding proteins suggests a methylase-preferred recognition sequence of Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe, with the COOH-terminal flanking glycine being obligatory. Taken together with data in the literature, identification of the sites of A1 arginine methylation strongly suggests a role for this modification in modulating the interaction of A1 with nucleic acids.

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.

A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.

Identification of the active site nucleophile in nucleoside 2-deoxyribosyltransferase as glutamic acid 98.

2'-Fluoro-2'-deoxyarabinonucleosides are time-dependent inhibitors of nucleoside 2-deoxyribosyltransferase. 2,6-Diamino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFDAP) inhibited the enzyme by formation of a primary complex (Kd = 140 microM) that isomerized to a secondary complex with a first-order rate constant of 0.2 min-1. Inhibited enzyme contained stoichiometric amounts of covalently bound 2'-fluoro-2'-deoxyarabinosyl moiety, recovered less than 5% of its activity after storage for a week at 5 degrees C, but regained over 70% of the lost activity by treatment with 600 microM Ade. 6-Amino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFAdo) was a product of the reactivation reaction. Proteolysis of inhibited enzyme identified a modified fragment that spanned residues 82-107 which could not be sequenced past Gly-96. dFDAP-inhibited enzyme and enzyme reacted with normal substrates (i.e. dThd and dAdo) were hydrolyzed between Met-97 and Glu-98 by 0.1 M NaOH. These findings and model studies on the base lability of peptides containing glutamyl esters suggested that the gamma-carboxylate of Glu-98 was esterfied during catalysis. The role of Glu-98 was confirmed by changing this residue to alanine. The specific activity of wild-type enzyme was 3 orders of magnitude greater than that of the mutant enzyme. Collectively, chemical modification and mutagenesis studies have identified Glu-98 as the active site nucleophile of nucleoside 2-deoxyribosyltransferase.

Purification and characterization of a rat liver enzyme that hydrolyzes valaciclovir, the L-valyl ester prodrug of acyclovir.

Valaciclovir is an oral prodrug of the antiherpetic agent acyclovir. An enzyme that hydrolyzes valaciclovir to acyclovir, valaciclovir hydrolase (VACVase), was purified from rat liver and characterized. VACVase was a basic (pI 9.4) protein associated with mitochondria. It was monomeric and had a molecular mass of 29 kDa. Amino acid sequences of six VACVase peptides, including its NH2 terminus (13 amino acids) and accounting for approximately 20% of its complete sequence, were not found in the SwissProt protein data base. VACVase hydrolyzed other amino acid esters of acyclovir in addition to valaciclovir (kcat/Km = 58 mM-1 s-1), with a preference for the L-alanyl (kcat/Km = 226 mM-1 s-1) and L-methionyl (kcat/Km = 200 mM-1 s-1) esters. It did not hydrolyze other types of esters or numerous di- and tripeptides and aminoacyl-beta-naphthylamides. Hydrolysis of valaciclovir by VACVase was not inhibited by amastatin, antipain, aprotinin, bestatin, chymostatin, E-64, EDTA, ebelactone A, ebelactone B, elastatinal, leupeptin, pepstatin, or phosphoramidon. It was neither inhibited nor activated by Ca2+, Co2+, Mg2+, Mn2+, or Zn2+. Therefore, this enzyme is not a typical esterase or peptidase and, to our knowledge, it has not been described previously. Its physiological function is not known; however, it may play a significant role in the biotransformation of valaciclovir to acyclovir.

Purification and characterization of the constitutive nitric oxide synthase from human placenta.

Human endothelial nitric oxide synthase (NOS) mRNA was detected in human placenta. In contrast, mRNAs for human neuronal NOS or for human inducible NOS were not detected in placenta. Subsequently, NOS was purified over 3800-fold from placental extract to greater than 80% homogeneity. A single band with an apparent molecular weight of 135 kDa was identified by [125I] calmodulin binding to proteins in a sodium dodecyl sulfate-polyacrylamide gel, which is consistent with the predicted size of the endothelial NOS. Furthermore, the sequence of eight internal peptides derived from this 135-kDa protein was identical to the published sequence of human endothelial NOS. As has been shown for all constitutive NOS isozymes, the purified NOS was absolutely dependent on calcium and calmodulin. NOS was also purified from human umbilical vein endothelial cells and, on the basis of similar kinetic parameters and dependence upon calcium and calmodulin, appeared to be the same as the purified placental NOS. Together, these data indicate that the placental NOS is the constitutive NOS isozyme from endothelial tissue.

Novel peptides from adrenomedullary chromaffin vesicles.

The adrenal medulla chromaffin vesicle (CV) contains, on a weight basis, as much soluble protein and peptide as catecholamine. The bulk of the protein is accounted for by chromogranins (Cgr) A, B and C. Additionally, a large variety of neuropeptides and their precursor proteins have been found recently within these vesicles. Nevertheless, fractionation of CV lysates indicates the presence of many more peptides than previously reported. In the hope of finding novel bioactive peptides, we initiated a systematic isolation and characterisation of CV peptides. Bovine CV pellets were prepared by sucrose gradient centrifugation and immediately boiled in water to avoid degradation of native proteins and peptides. The water lysates were fractionated through a battery of reversed-phase and ion-exchange high-performance chromatographic steps. We fully or partially characterised a substantial number of novel peptides derived from CgrA and CgrB. A tetradecapeptide and a 13 kDa extended peptide were derived from the bovine homologue of rat secretogranin III. Peptides corresponding to C-terminal fragments of 7B2 and proteoglycan II were also found. Additionally, several sequences had no known precursors. Of the sequences derived from known precursors some corresponded to fragments bracketed by pairs of basic amino acids, but others were preceded or followed by single basic residues or by unusual putative cleavage sites. Some of these peptides were postranslationally modified (pyroglutamylation, glycosylation, phosphorylation, amidation). A significant degree of structural conservation of some of these peptides across species suggests that they may exert biological effects when cosecreted with catecholamines during splanchnic stimulation.

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

Identification of a 7B2-derived tridecapeptide from bovine adrenal medulla chromaffin vesicles.

1. A novel tridecapeptide was isolated from extracts of bovine adrenal medulla chromaffin vesicles and the primary structure determined to be SVPHFSDEDKDPE. 2. This peptide is identical to the C termini of human and porcine 7B2 and is highly homologous to the same region of the mouse and Xenopus lavis protein. 3. In all these species the homologous peptide is preceded by a pair of lysine residues, a potential proteolytic processing site. 4. Ser6 is part of a well-conserved casein kinase II consensus phosphorylation sequence. Evidence for phosphorylation of this residue was obtained during Edman sequencing. 5. Thus, this novel adrenal medullary probably arises from the posttranslational processing of the bovine 7B2 protein.

Proteolysis at the secretase and amyloidogenic cleavage sites of the beta-amyloid precursor protein by acetylcholinesterase and butyrylcholinesterase using model peptide substrates.

1. It was recently proposed that acetylcholinesterase (AChE), in addition to its esteratic activity, has proteolytic activity such that it may cleave the beta-amyloid precursor (beta-APP) within the beta-amyloid sequence. The purpose of this paper was to examine further whether AChE or butyrylcholinesterase (BuChE) had associated proteinase activity that was involved in the metabolism of beta-APP. 2. The ability of various preparations of AChE and BuChE to hydrolyze two synthetic fragments of beta-APP695 as model substrates containing the normal and aberrant cleavage sites was studied. 3. Digestion of these synthetic substrates with commercial preparations of Electrophorus electricus AChE indicated the presence of a trypsin-like proteolytic activity cleaving each peptide at the carboxy-terminal side of an internal lysine residue. 4. Purification of the trypsin-like proteinase activity by aminobenzamidine affinity chromatography yielded a preparation that was devoid of AChE activity but retained all of the proteinase activity. 5. Amino-terminal sequence analysis of this preparation showed that the first 13 amino acid residues were identical to beta-pancreatic trypsin. 6. These data indicate that the proteinase activity found in these commercial preparations of AChE is due to contamination with trypsin.

Site and consequences of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase type "Gr".

CaM kinase-Gr is a Ca2+/calmodulin-dependent protein kinase that is enriched in brain and thymus. The enzyme was isolated from rat cerebellum, which contained alpha (M(r) 65,000) and beta (M(r) 67,000) polypeptides, and rat forebrain, which contained only the alpha polypeptide. Both enzyme preparations readily underwent autophosphorylation with dramatic up-regulation of their Ca2+/calmodulin-dependent, as well as-independent, activity. Autophosphorylation also caused a characteristic retardation in the electrophoretic gel mobility of the alpha and beta polypeptides. Treatment of autophosphorylated CaM kinase-Gr with acid phosphatase fully dephosphorylated the enzyme and reversed the changes in electrophoretic migration of both polypeptides. Phosphopeptide mapping indicated that the alpha and beta polypeptides were phosphorylated on identical or homologous sites, which probably induces similar structural and catalytic modifications in the two polypeptides. The actual site(s) of autophosphorylation was determined by the purification and amino acid sequencing of tryptic peptides from 32P-labeled CaM kinase-Gr. The major site of autophosphorylation was localized to a novel N-terminal domain, which is rich in Ser/Thr/Pro residues. The functional and structural studies on CaM kinase-Gr autophosphorylation imply that the enzyme is comprised of two regulatory domains, one on either side of a catalytic domain, followed by a C-terminal, putative association domain. The properties of such a structural model are discussed.

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.

Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil.

Uracil analogues with appropriate substituents at the 5-position inactivated dihydropyrimidine dehydrogenase (DHPDHase). The efficiency of these inactivators was highly dependent on the size of the 5-substituent. For example, 5-ethynyluracil inactivated DHPDHase with an efficiency (kinact/Ki) that was 500-fold greater than that for 5-propynyluracil. 5-Ethynyluracil inactivated DHPDHase by initially forming a reversible complex with a Ki of 1.6 +/- 0.2 microM. This initial complex yielded inactivated enzyme with a rate constant of 20 +/- 2 min-1 (kinact). Thymine competitively decreased the apparent rate constant for inactivation of DHPDHase by 5-ethynyluracil. The absorbance spectrum of 5-ethylnyluracil-inactivated DHPDHase was different from that of reduced enzyme. These optical changes were correlated with the loss of enzymatic activity. 5-Ethynyluracil inactivated DHPDHase with a stoichiometry of 0.9 mol of inactivator per mol of active site. Enzyme inactivated with [2-14C]5-ethynyluracil retained all of the radiolabel after denaturation in 8 M urea, but lost radiolabel under acidic conditions. These results suggested that inactivation was due to covalent modification of an amino acid residue and not due to modification of a noncovalently bound prosthetic group. A radiolabeled peptide was isolated from a tryptic digest of the enzyme inactivated with [2-14C]5-ethynyluracil. The sequence of this peptide was Lys-Ala-Glu-Ala-Ser-Gly-Ala-Y-Ala-Leu-Glu-Leu-Asn-Leu-Ser-X-Pro-His-Gly- Met-Gly-Glu-Arg, where X and Y were unidentified amino acids. Since the radiolabel was lost from the peptide during the first cycle on the amino acid sequenator, the position of the radiolabeled amino acid was not determined. The amino acid residue designated by X was identified as a cysteine from previous work with DHPDHase inactivated with 5-iodouracil. In contrast to 5-ethynyluracil, 5-cyanouracil was a reversible inactivator of the enzyme. 5-Cyanouracil-inactivated enzyme slowly regained activity (t1/2 = 1.8 min) after dilution into the standard assay. DHPDHases isolated from rat, mouse, and human liver had similar sensitivities to inactivation by 5-alkynyluracils.

Inactivation of dihydropyrimidine dehydrogenase by 5-iodouracil.

5-Iodouracil was a substrate for bovine liver dihydropyrimidine dehydrogenase (DHPDHase) and was a potent inactivator of the enzyme. NADPH increased the rate of inactivation and thymine protected against inactivation. These findings suggest that 5-iodouracil was a mechanism-based inactivator. However, dithiothreitol and excess 5-iodouracil protected the enzyme against inactivation. Thus, a reactive product, presumably 5-iodo-5,6-dihydrouracil generated through the enzymatic reduction of 5-iodouracil, was released from DHPDHase during processing of 5-iodouracil. Since only 18% of [6-3H]5-iodouracil reduced by DHPDHase was covalently bound to the enzyme and radiolabel was not lost to the solvent as tritium, the partition coefficient for inactivation was 4.5. However, the enzymatic activity was completely titrated with 1.7 mol of 5-iodouracil per mol of enzyme-bound flavin. These results indicate that there was 0.31 mol of enzyme-bound inactivator per mol of enzyme flavin. This suggests there were 3.2 flavins per active site, which is consistent with the report of multiple flavins per enzymic subunit (Podschun, B., Wahler, G., and Schnackerz, K. D. (1989) Eur. J. Biochem. 185, 219-224). DHPDHase was inactivated by 2.1 mol of racemic 5-iodo-5,6-dihydrouracil per mol of active sites. The stoichiometry for inactivation of the enzyme by the nonenzymatically generated enantiomer of 5-iodo-5,6-dihydrouracil was calculated to be 1. Two radiolabeled fragments were isolated from a tryptic digest of DHPDHase inactivated with radiolabeled 5-iodouracil. The amino acid sequences of these peptides were Asn-Leu-Ser-X-Pro-His and Asn-Leu-Ser-X-Pro-His-Gly-Met-Gly-Glu-Arg where X was the modified amino acid containing radiolabel from [6-3H]5-iodouracil. Fast atom bombardment mass spectral analysis of the smaller peptide yielded a protonated parent ion mass of 782 daltons that was consistent with X being a S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteinyl residue.

Relationship of genes encoding Ca2+/calmodulin-dependent protein kinase Gr and calspermin: a gene within a gene.

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells (CaM kinase Gr) is a neuronal calmodulin-dependent protein kinase whose purification and partial cloning from rat brain has been described. A combination of the polymerase chain reaction and cDNA library screening was used to determine the DNA sequence that encodes most of the remaining polypeptide sequence. The deduced amino acid sequence was confirmed by comparison with the peptide sequence from purified CaM kinase Gr. Analysis of this sequence indicated the presence of potential catalytic, regulatory, and association domains with 42% overall homology to the alpha subunit of another neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase II. The degree of homology within the catalytic domain was 58% with conservation of all invariant amino acids. The portion of sequence that extended from the hypothesized calmodulin-binding domain to the carboxyl terminus of the protein was identical at both the amino acid and nucleotide level to the noncatalytic, calmodulin-binding protein calspermin from rat testis. Screening a genomic library with a portion of the cDNA for CaM kinase Gr allowed the isolation of a genomic clone that contained at least 9 kilobases (kb) of the gene for CaM kinase Gr. Analysis of the sequence revealed that the coding sequences for calspermin were contained within the CaM kinase Gr gene and that alternative splicing of internal exons may lead to the formation of the two different proteins, CaM kinase Gr and calspermin.

Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions.

The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel tetradecapeptide isolated from bovine adrenal medulla chromaffin vesicles with strong homology to an internal sequence coded by the rat 1B1075 (Preprosecretogranin III) gene.

A novel tetradecapeptide, PheProLysProAlaGlySerGlnAspLysProLeuHisAsn, was isolated from boiling water extracts of bovine adrenal medulla chromaffin vesicles. The primary structure of the peptide was characterized by amino acid analysis, fast atom bombardment mass spectrometry, and gas-phase sequencing. The synthetic and native peptides comigrated on reversed-phase high-performance liquid chromatography, supporting the proposed sequence. This peptide shares 86% homology to residues 67-80 of the recently reported rat 1B1075 gene product secretogranin III and probably represents a processed product derived from the bovine equivalent.

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain.

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.

Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles.

Partial acid cleavage, comparative HPLC tryptic peptide mapping and amino acid sequencing of the C1 and C2 proteins of HeLa heterogeneous nuclear ribonucleoprotein (hnRNP) particles demonstrate that proteins C1 and C2 differ in primary structure by the presence of a 13 amino acid insert sequence in C2. This C2 insert sequence occurs after either glycine 106 or serine 107 in C1. The additional 13 amino acids that are present in C2 account for the observed molecular weight difference between the C1 and C2 hnRNP proteins on SDS polyacrylamide gel electrophoresis. Because C1 and C2 appear identical except for the 13 residue insert and because the 3' and 5' untranslated regions of the corresponding mRNAs also appear to be the same (Swanson et al., Mol. Cell. Biol. 7: 1731-1739), it is possible that both polypeptides are produced from a single transcription unit through an alternative splicing mechanism.