Locating peptides generated by limited proteolysis in known protein sequences by means of computer search

A computational method is presented for locating peptides on known protein sequences using their molecular weights (estimated by SDS polyacrylamide gel electrophoresis) and their composition (obtained by amino acid analysis from eluted gel bands). The technique is easy and rapid, and appears particularly valuable if recoveries of peptides are low, or if they are heterogeneous due to secondary proteolytic degradation. An analysis of limited proteolysis of maltoporin (Schenkman, S. et al. [1984] J. Biol. Chem. 259, 7570-7576) is used to illustrate applicability and reliability of the method, which can also be applied to confirm gene-derived sequences.     

Purification and complete primary structures of the heparin-, cell-, and DNA-binding domains of bovine plasma fibronectin

The complete amino acid sequences of the heparin-, cell- and DNA-binding domains of bovine plasma fibronectin have been determined. The fragments were generated from the 170-kDa central plasmic fragment by extensive digestion with chymotrypsin, and they contain 268, 300 and 269 amino acid residues, respectively. No half-cystines or cysteines were found in these sequences. A glucosamine-based oligosaccharide group is attached to Asn-108 in the sequence of the DNA-binding domain. Only one of the three types of internal homology found in fibronectin [Petersen et al. (1983) Proc. Natl Acad. Sci. USA 80, 137-141], namely the type III homology, occurs in these three fragments, and each of them consists of approximately three stretches of this type III homology. Part of the arrangement of peptides was derived by comparison with the partial cDNA sequence for human fibronectin recently reported [Kornblihtt et al. (1984) Nucleic Acids Res. 12, 5853-5868].     

Evidence for the extent of insertion of the active site loop of intact alpha 1 proteinase inhibitor in beta-sheet A.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [L?bermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.     

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria. Reply to a commentary

Costa, L.E., Reynafarje, B. and Lehninger, A.L. [(1984) J. Biol. Chem. 259, 4802-4811] have reported 'second-generation' measurements of the H+/O ratio approaching 8.0 for vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria. In a Commentary in this Journal [Krab, K., Soos, J. and Wikstr?m, M. (1984) FEBS Lett. 178, 187-192] it was concluded that the measurements of Costa et al. significantly overestimated the true H+/O stoichiometry. It is shown here that the mathematical simulation on which Krab et al. based this claim is faulty and that data reported by Costa et al. had already excluded the criticism advanced by Krab et al. Also reported are new data, obtained under conditions in which the arguments of Krab et al. are irrelevant, which confirm that the H+/O ratio for succinate oxidation extrapolated to level flow is close to 8.     

Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta

We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.     

Conformational study of endothelins and sarafotoxins with the cystine-stabilized helical motif by means of CD spectra.

A series of CD measurements were carried out on members of peptides in the endothelin and sarafotoxin families. The helical structures taken by these peptides containing the helical motif with the sequences of Cys-X-X-X-Cys and Cys-X-Cys [Y. Kobayashi et al. (1991) Neurochemistry International Vol. 18, pp. 525-534] are classified into three groups: a group of structures of ET-1, ET-2 and vasoactive intestinal contractor (VIC), a group of sarafotoxin, and a group of ET-3.     

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.     

Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase

A series of tripeptides that satisfy the -Asn-Xaa-Thr/Ser- primary sequence requirement [Marshall, R. D. (1972) Annu. Rev. Biochem. 41, 673-702] for N-glycosylation have been synthesized and examined as potential acceptors in an oligosaccharyltransferase assay. Of these, six (Ac-Asn-Ala-Thr-NH2, Ac-Asn-Leu-Thr-NH2, Ac-Asn-Asp-Thr-NH2, Ac-Asn-D-Ala-Thr-NH2, Ac-Asn-Pro-Thr-NH2, and Ac-Asn-AIB-Thr-NH2) were examined for solution conformational properties in dimethyl sulfoxide with use of amide proton temperature coefficients, 3JHN alpha analysis [Pardi, A., et al. (1984) J. Mol. Biol. 180, 741-751], and 2-D ROESY experiments [Bothner-By, A. A., et al. (1984) J. Am. Chem. Soc. 106, 811-813]. The analysis reveals that the peptides that serve as acceptors in the transferase assay demonstrate similar conformational properties in solution. These are highlighted by a secondary structural motif that involves the interaction between the asparagine side-chain carboxamide and the backbone amide of the threonine. The peptides that show very poor acceptor, or even nonacceptor, properties in the oligosaccharyltransferase assay demonstrate different conformational features in solution. These observations may explain the distinct biological activity observed for these peptides.     

Elucidation of the primary structures of the cockroach hyperglycaemic hormones I and II using enzymatic techniques and gas-phase sequencing

ABSTRACT. Two peptides, HGHI and HGHII, both inducing hyperglycaemia and activation of fat body glycogen phosphorylase can be isolated from the corpora cardiaca of the American cockroach, Periplaneta americana , using high-performance liquid chromatography. Both peptides are N-terminally blocked by a pyroglutamate residue and are thus not available for sequencing methods using the Edman degradation as this technique requires a free N-terminus. The blocked peptides were treated with pyroglutamate aminopeptidase to cleave the pyroglutamate residue, and the C-terminus of each peptide is also blocked and neither molecule can be cleaved by carboxypeptidase A. The following sequences for hyperglycaemic hormones HGHI and HGHII have been revealed using gas-phase sequencing.
HGHI pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂
HGHII pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂
Both peptides show remarkable similarities to locust adipokinetic hormones I and II and prawn red-pigment concentrating hormone, and are identical to two myotropic peptides MI and MII (O'Shea et al. , 1984; Witten et al. , 1984) and two cardioactive and hyperglycaemic peptides CC-1 and CC-2 (Scarborough et al. , 1984), respectively, also isolated from the corpora cardiaca of P. americana.     

Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.

Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.     

A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].     

A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155–9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [ Science 230 (1985) 1350–1353] and results in a more than 10⁴-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 μg of tumor DNA.     

Profilin-actin complexes directly elongate actin filaments at the barbed end.

We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. G., et al. (1983) J. Cell Biol. 97, 112-124] and demonstrated by Pollard and Cooper [(1984) Biochemistry 23, 6631-6641] by electron microscopy. We show that a model without any outside energy supply, in contrast to the mechanism proposed by Pollard and Cooper, can be fitted to our and their [Kaiser et al. (1986) J. Cell Biol. 102, 221-226] findings. Input of outside energy is necessary only if profilin-mediated elongation continues after free G-actin has been lowered to or below the critical concentration observed at the barbed end in the absence of profilin.     

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.     

Use of Bromodeoxyuridine For Cell Kinetic Studies In Intact Animals

Abstract. A method is described for the use of BUdR for tracing cell proliferation patterns in the intestinal mucosa of intact mice.
The method has several distinct advantages over existing methods.
Bromodeoxyuridine (BUdR) is a well-established alternative to tritiated thymidine ([³H]TdR) as a tracer for studying DNA replication. However, its use in cytological as opposed to biochemical studies has been largely confined to examination of metaphase spreads, particularly analysis of sister chromatid exchange (Block, 1982). For this, BUdR incorporation into DNA has been demonstrated using the fluorescent dye Hoechst 33258, together with fluorescence microscopy (Latt, 1973), or Giemsa staining (Perry & Wolf, 1974). Recently, introduction of a monoclonal antibody which recognizes BUdR in single-stranded DNA (Gratzner, 1982) has enabled BUdR to be used for studying cell cycle kinetics in a manner exactly analogous to the use of [³H]TdR. This has been reported for whole cells in suspension and in monolayer (Dolbeare et al. , 1983; Dean et al. , 1984; Raza et al. , 1984). BUdR included in tissue culture medium is taken up and incorporated into newly synthesized DNA via the same pyrimidine salvage pathway as [³H]TdR (thymidine kinase). A concentration of as little as 10 μm—well below cytotoxic levels (Cerni, 1984)—is sufficient to give readily detectable labelling by immunocytochemistry with a pulse of less than 15 min. the validity of BUdR labelling for cell kinetic studies has been well established in comparisons with other methods by Dolbeare et al. (1983), Dean et al. (1984), and Raza et al. (1984).
We describe here the use of BUdR together with an immunocytochemical detection system applied to sections of wax-embedded tissues, which provides a convenient method of cell cycle analysis in intact animals.     

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.