Amino-acid sequence of toxin III of Naja haje.

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja nivea alpha (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).     

Primary structure of protein S13 from the small subunit of escherichia coli ribosomes.

The experimental details which led to the determination of the complete primary structure of protein S13 from the small subunit of Escherichia coli ribosomes are presented. S13 consists of 117 amino acid residues and has the following composition: Asp6, Asn2, Thr6, Ser6, Glu6, Gln2, Pro4, Gly11, Ala11, Cys1, Val7, Met2, Ile12, Leu9, Tyr2, Phe1, His3, Lys11 and Arg15. Tryptophan was not found. The molecular weight of protein S13 as derived from the sequence shown in Fig. 1 is 12970. The amino acid sequence of the protein was determined by combining the results obtained from liquid phase Edman degradation of the intact protein with those from the peptides isolated after enzymatic digestions with trypsin, Staphylococcus aureus protease and thermolysin. Additional information about the primary structure was derived from analysis of the chymotryptic peptides of protein S13 and from its digestion with carboxypeptidase C. The amino acid sequence of protein S13 was compared with the published sequences of the other ribosomal proteins of E. coli and predictions for the secondary structure of this protein were made.     

The primary structure of Bacillus subtilis acidic ribonsomal protein B-19. Isolation and characterization of peptides and the complete amino acid sequence

The complete primary structure of Bacillus subtilis acidic protein B-L9, functionally equivalent to protein L7/L12 from E. coli, has been determined. B-L9 is composed of 122 residues and has the amino acid composition: Asp3, ASN3, Thr4, Ser3, Glu22, Gln1, Pro3, Gly11, Ala21, Val14, Ile9, Leu12, Phe2, Lys13, and Arg1. The molecular weight of B-L9 is 12,633. The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a modified Beckman sequenator, and micro dansyl-Edman degradation of the peptides obtained from digestions with trypsin, thermolysin, Staphylococcus aureus protease, chymotrypsin and pepsin. A comparison of protein B-L9 from B. subtilis with E-L12 from E. coli shows a relatively high degree of homology.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

Characterization of an alkaline subtilopeptidase type Pfizer.

The physiochemical properties, amino acid composition and profile of the the tryptic peptides for an alkaline subtilopeptidase type Pfizer have been determined. The enzyme is stable in the pH range from 5 to 10, has a pH optimum of 9.5 to 10, and is relatively stable for a period of 2 h up to a temperature of 50C. Homogeneity was demonstrated by electrophoretic techniques and the mobilities indicated on isoelectric point of 8.7. The molecular weight was found to be 25,000 by gel filtration. The amino acid composition was found to be Ala32, Arg4, Aspgamma8, Glu15, Gly29, His4, Ile9, Leu13, Lys11, Met5, Phe4, Pro14, Ser31, Thr17, Tyr9, Val22, a total of 247 amino acid residues. The enzyme does not contain either disulfide bonds or cysteine, and lacks tryptophan as well. The N-terminal end-group residue is alanine: the C-terminal amino acid is arginine. Tryptic hydrolysis of the enzyme produced 15 peptides which were separated by gradient elution on Dowex 50-X2. The amino acid composition of each appropriately purified tryptic peptide was established.     

Snake venom toxins. The primary structure of protein S4C11. A neurotoxin homologue from the venom of forest cobra (Naja melanoleuca).

Six minor protein constituents (S4C10-S4C15) have been isolated from the venom of Naja melanoleuca. The complete amino acid sequence of S4C11 has been established and indicates that it is a homologue of the neurotoxins which are found in elapid venoms. The other proteins appear from the amino acid compositions to be homologues of the cyto- or cardiotoxins found in cobra venoms. Protein S4C11 has a low toxicity, failing to kill mice at an intravenous dose of 20 mug/g body weight. The sequence of the first 25 residues out of the total of 65, was determined using the automatic sequenator. The remainder of the sequence was derived with the aid of tryptic and chymotryptic peptides. The sequence showed the unusual feature of having 65 amino acid residues including 10 half-cystine residues.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

The core protein of alphaviruses. 1. Purification of peptides and complete amino-acid sequence of Semliki Forest virus core protein

The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiments is in agreement with the sequence predicted from the nucleotide sequence of the mRNA [Garoff et al. (1980) Proc. Natl Acad. Sci. USA, 77, 6376-6380]. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition: Asp12, Asn9, Thr16, Ser10, Glu11, Gln15, Pro23, Gly20, Ala23, Val19, Met8, Ile11, Leu9, Tyr7, Phe6, His7, Lys37, Arg15, Trp5, Cys4, and an Mr of 29919. It contains 22.1% basic amino acids, mainly lysines, compared with a total of 8.6% acidic residues. The resulting surplus of positive charge is located in the N-terminal half of the protein (predominantly arginines at positions 12-21 and lysines at positions 66-114). Other amino acids are also unevenly distributed; proline and glutamine are accumulated in the N-terminal half of the sequence whereas histidine, glycine and the acidic residues are mainly present in the C-terminal part. This distribution suggests that the virus core protein consists of two or more structural domains.     

Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles

The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH(+)(4), while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradation.     

The primary structure of rat liver ribosomal protein L39

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.     

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

The primary sequence of chicken myoglobin (Gallus gallus).

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.     

The amino acid sequence of Escherichia coli cyanase

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.     

Complete amino acid sequence of the major component myoglobin of finback whale (Balaenoptera physalus)

The complete amino acid sequence of the major component myoglobin from finback whale, Balaenoptera physalus, was determined by the automated Edman degradation of several large peptides obtained by specific cleavages of the protein. Three easily separable peptides were obtained by cleaving with cyanogen bromide at the two methionine residues and one large peptide was isolated after cleavage with (2-p-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. More than 60% of the covalent structure was established by the sequential degradation of three of these peptides and the apomyoglobin. An additional 30% of the primary sequence was established with peptides obtained from tryptic digestion of both the apomyoglobin and the acetimidoapomyoglobin, and the final 10% of the sequence was completed after digestion of the two larger cyanogen bromide peptides with S. aureus strain V8 protease. This myoglobin differs from that of the sperm whale, Physeter catodon, at 15 positions, from that of the arctic minke whale, Balaenoptera acutorostrata, at 3 positions, and from that of the California gray whale, Eschrichtius gibbosus, at 4 positions. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

ATP-dependent Saccharomyces cerevisiae phospho enol pyruvate carboxykinase: isolation and sequence of a peptide containing a highly reactive cysteine.

Saccharomyces cerevisiae phospho enol pyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per mol of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCl2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphoryl-binding sequence proposed for some nucleotide-binding proteins.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

---

---

Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.