The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin.
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Adenosine Monophosphate↗
Biomedical subjects
R L Heinrikson
Publications and source records attributed to R L Heinrikson.
Platelet factor 4 is chemotactic for neutrophils and monocytes.
Platelet factor 4 is shown to be a chemotactic protein for human polymorphonuclear leukocytes and monocytes at concentrations found in human serum and reached locally in injured tissue. The maximum chemotactic response to platelet factor 4 nearly equals that achieved with saturating concentrations of the chemotactic activity derived from the fifth component of human complement, C5. Cells desensitized to C5 chemotactic activity retain chemotactic responsiveness to platelet factor 4. Serum contains inhibitory capacity against the chemotactic activity associated with platelet factor 4. Our results suggest that the local release of platelet factor 4 may be an important stimulus attracting inflammatory cells to sites of blood vessel injury.
Blood Coagulation Factors↗
Phosphorylation of rat hepatic fructose-1,6-bisphosphatase and pyruvate kinase.
Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Brief exposure of the 32P-labeled enzyme to trypsin removed all radioactivity from the enzyme core and produced a single-labeled peptide. The partial sequence of the 17-amino acid peptide was found to be Ser-Arg-Pro-Ser(P)-Leu-Pro-Leu-Pro-(Ser2, Glx2, Pro2, Leu, Arg2). The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of native fructose bisphosphatase were compared with those of rat liver type L pyruvate kinase where the sequence around the phosphoserine is known (Arg-Arg-Ala-Ser(P)-Val; Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563). The Km for pyruvate kinase (17 microM) was less than that for fructose bisphosphatase (58 microM); the Vmax was about 3-fold greater with pyruvate kinase as substrate. The relationship between the rates of phosphorylation of these native substrates and the amino acid sequences surrounding the phosphorylated sites is discussed.
Amino Acids↗
Amino acid sequence at the allosteric site of sheep heart phosphofructokinase.
Covalent modification of sheep heart phosphofructokinase with the affinity labeling reagent p-fluorosulfonyl[14C]benzoyl-5'-adenosine caused a loss of allosteric properties. This modification appears to occur at the binding site that is specific for the allosteric activators AMP, cAMP, and ADP (Mansour, T.E., and Colman, R.F. (1978) Biochem. Biophys. Res. Commun. 81, 1370-1376). In the current study, the site of modification has been demonstrated to be a lysine residue. A nonapeptide containing a covalently bound [14C]carboxybenzenesulfonyl group attached to alysine residue has been isolated following tryptic digestion. The amino acid sequence of the peptide is Asn-Phe-Ala-Thr-Lys-Met-Gly-Ala-Lys. The fifth residue in this sequence, lysine, contained the covalently bonded reagent.
Allosteric Site↗
The covalent structure of bovine liver rhodanese. NH2-terminal sequence and partial structural analysis of tryptic peptides from the citraconylated protein.
Nineteen tryptic peptides produced by cleavage at 18 of the 20 arginyl residues in citraconylated S-carboxymethylcysteinyl-rhodanese have been isolated by a combination of gel filtration and high voltage paper electrophoresis. These Tc fragments account for all of the 293 residues in the parent polypeptide and their partial or complete sequences have been determined by automated and manual Edman degradation. In some cases, sequence analyses were completed by degradation of peptides derived by secondary cleavages of the decitraconylated Tc fragments with trypsin, chymotrypsin, or the protease from Staphylococcus aureus. Automated Edman degradation of intact S-carboxymethylcysteinyl-rhodanese was performed for 60 cycles; the information thus obtained permitted the alignment of seven of the Tc fragments and gave the sequence of the first 79 residues in the polypeptide chain. The Tc peptide at the COOH terminus of rhodanese was placed by virtue of the fact that it contained no arginine. Structural analysis of the Tc peptides provided the sequences surrounding all five of the methionyl residues in the enzyme. One of the methionines was found in a 19-residue Tc fragment which also contained the cysteinyl residue essential for catalysis.
Amino Acid Sequence↗
The covalent structure of bovine liver rhodanese. Isolation and partial structural analysis of cyanogen bromide fragements and the complete sequence of the enzyme.
Cyanogen bromide fragments from reduced and carboxymethylated rhodanese have been isolated by gel filtration and ion exchange chromatography on columns of Sephadex G-50 and sulfoethyl-Sephadex C-25, respectively. Partial or complete structural analysis of these fragments has permitted the ordering in sequence of all eleven of the nonaligned tryptic peptides from citraconylated, S-carboxymethylcysteinyl-rhodanese and has thus provided the complete covalent structure of the enzyme. Rhodanese is a single polypeptide of 293 residues and the molecule weight calculated from the covalent structural analysis is about 32,900. The cysteinyl residue implicated in the catalytic function of rhodanese is at position 247. In some preparations of the enzyme the NH2-terminal dipeptide Val-His is missing and the sequence begins with the glutamine at position 3. The rhodanese thus obtained contains 291 amino acid residues and possesses full enzymic activity. X-ray crystallographic analysis of rhodanese has shown that the halves of the molecule (Domains I and II) are nearly identical in conformation. Comparative analysis of the sequences in Domains I and II containing residues with conformationally equivalent alpha C atoms has revealed some degree of homology between the halves of the rhodanese polypeptide. Nethertheless, the structural equivalence of the rhodanese domains is reflected much more by their similarity in tertiary structural than by their sequence homology, even when the sequence comparisons are optimized with reference to the crystallographic results.
Amino Acid Sequence↗
Active site cysteinyl and arginyl residues of rhodanese. A novel formation of disulfide bonds in the active site promoted by phenylglyoxal.
Chemical modification studies of bovine liver rhodanese have underscored important distinctions between free rhodanese and the catalytic intermediate in which the sulfane atom of the sulfur donor is bound covalently to the enzyme (sulfur-rhodanese). Treatment of free rhodanese with near-stoichiometric quantities of either iodoacetate or phenylglyoxal results in the rapid modification of the essential sulfhydryl group of Cys-247 and the consequent inactivation of the enzyme. Analysis of rate data for the iodoacetate reaction showed that the apparent pK of this group is 7.8 in free rhodanese and 6.7 to 7.0 in complexes of the enzyme with analogs of sulfur donor substrates, in agreement with the previous inference from steady state kinetic observations. Inactivation of free rhodanese by phenylglyoxal in the presence of cyanide was shown to be caused by a novel reaction in which disulfide bonds are formed between Cys-247 and either Cys-254 or Cys-263. In contrast to these results with free rhodanese, the sulfur-substituted enzyme is not inactivated by iodoacetate and is only relatively slowly inactivted by treatment with substantial excesses of phenylglyoxal. The loss of enzyme activity in sulfur-rhodanese does not involve cysteinyl residues but can be correlated with the modification of guanidino groups, notably that of Arg-186, the side chain of which may play a role in substrate binding. These chemical modification studies have implications with respect to the chemical mechanism of rhodanese catalysis and the interpretation of the x-ray crystallographic analysis of this enzyme.
Aldehydes↗
Postsynthetic modification of high mobility group proteins. Evidence that high mobility group proteins are acetylated.
High mobility group proteins were isolated from calf thymus and duck erythrocyte nuclei and the possibility was investigated that these proteins undergo acetylation similar to that occurring in some histones. Dinitrophenylation of the proteins followed by acid hydrolysis and amino acid analysis indicated that 2 to 3% of the lysine residues present were unavailable for reaction with fluorodinitrobenzene. Extensive enzymatic degradation with trypsin and pronase and subsequent amino acid analysis showed a significant amount of material eluting at the position of epsilon-N-acetyllysine. Recovery and acid hydrolysis of this material generated a peak eluting in the lysine position. In vitro radioactive labeling of calf thymus nuclei with [3H]acetate yielded labeled high mobility group proteins. All of these findings are in accord with the conclusion that high mobility group proteins are acetylated and that acetylation occurs as a postsynthetic modification of these proteins.
Acetylation↗
Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis.
The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.
Amino Acid Sequence↗
Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins.
The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.
Amino Acids↗
The covalent and tertiary structure of bovine liver rhodanese.
Bovine liver rhodanese is a single polypeptide of 293 amino acids in which the halves of the molecule assume analogous tertiary structures in the absence of substantial sequence homology. The sulphur atom transferred during catalysis is bound in persulphide linkage to Cys-247. Substrate binding seems to involve Arg-186 and Lys-249.
Amino Acid Sequence↗
Silkmoth chorion proteins. Their diversity, amino acid composition, and the NH-terminal sequence of one component.
Silkmoth eggshell (chorion) proteins have been characterized by electrophoresis on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels; up to 33 and 41 components, respectively, were detected from a single chorion. Some of these components are polymorphic, being absent from chorions of certain animals. A system of nomenclature for all chorion proteins is presented, based on their separation on sodium dodecyl sulfate and isoelectric focusing gels. The chorion is enriched in glycine, alanine, cysteine, and tyrosine and poor in methionine and histidine. The proteins were fractionated into four partially overlapping groups; all four are enriched in the above amino acids, although significant differences exist. Further fractionation by isoelectric focusing of one of the above groups, s/s, yielded seven components, two of which are homogeneous both on sodium dodecyl sulfate and isoelectric focusing gels. The amino acid compositions, molecular weights, and solubility properties of the components share certain features which distinguish s/s as a group from the other three groups. The sequence of the first 67 NH2-terminal residues of a homogeneous protein purified from s/s has been determined. The protein contains a cysteine-rich tail (3 cysteines in the first 18 residues) followed by a 49-residue segment which contains only a single cysteine residue. This latter segment also contains two different tetrapeptide sequences which are each repeated, one twice and the other four times.
Amino Acid Sequence↗
Covalent structural analysis of yeast inorganic pyrophosphatase.
The present paper describes the amino acid sequence analysis of the internal and COOH-terminal cyanogen bromide fragments of yeast inorganic pyrophosphatase (Sterner, R., Noyes, C., and Heinrikson, R.L. (1974) Biochemistry 13, 91-99). This information coupled with that derived from earlier structural studies of the enzyme (Sterner, R., AND Heinrikson, R.L. (1975) Arch. Biochem. Biophys. 165, 693-703) provides the complete covalent structure of the pyrophosphatase subunit. The majority of the sequence data was derived from automated Edman degradation of the intact cyanogen bromide fragments and the large tryptic peptides obtained from citraconylated derivates in which cleavages were restricted to arginyl residues. The structural determination was completed by analysis of tryptic and chymotryptic peptides from the decitraconylated fragments. The monomer peptide chain contains 285 amino acid residues and the molecular weight calculated from the sequence analysis is 32,042.
Amino Acid Sequence↗
Detection and kinetic behavior of preproinsulin in pancreatic islets.
Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.
Adenoma, Islet Cell↗
Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.
Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.
Amino Acid Sequence↗
Amino acid sequence of phospholipase A2-alpha from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants.
The complete amino acid sequence of Crotalus adamanteus venom phospholipase A2-alpha has been determined by analysis of the five tryptic peptides from the citraconylated, reduced, and S-[14C]carboxamidomethylated enzyme. Earlier studies (Tsao, F. H. C., Keim, P. S., and Heinrikson, R. L. (1975) Arch. Biochem. Biophys. 167, 706) provided the information necessary to align the tryptic fragments so that secondary cleavage procedures to establish overlaps were unnecessary. The subunit in the phospholipase A2-alpha dimer is a single polypeptide chain containing 122 amino acids and seven disulfide bonds. The histidine residue implicated in the active site of mammalian phospholipases is at position 47 in the C. adamanteus enzyme and is located in a domain of the molecule which is highly homologous in sequence with corresponding regions of phospholipases from a variety of venom and pancreatic sources. Comparative sequence analysis has revealed insights with regard to the function and evolution of phospholipases A2. Primary structural relationships observed among the snake venom enzymes parallel the phylogenetic classification of the venomous reptiles from which they were derived. It is proposed that phospholipases A2 of this general type be divided into two groups depending upon the presence or absence of distinctive structural features elucidated in this study.
Amino Acid Sequence↗
Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.
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Amino Acids↗
Applications of thermolysin in protein structural analysis.
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Amino Acid Sequence↗