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R L Heinrikson

Publications and source records attributed to R L Heinrikson.

At least 109 records · Page 6Linked to original sources
PubMed1985

Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme.

Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.

Adenosine Diphosphate↗
PubMed1984

A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.

Amino Acid Sequence↗
PubMed1984

The covalent protein structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Manduca sexta L.

The amino acid sequence has been determined for the insecticyanin from the hemolymph of the fifth instar larvae of the tobacco hornworm, Manduca sexta. The apoprotein is a single polypeptide chain of 189 amino acids, molecular weight 21,378, containing two disulfide bridges, 9-119 and 42-176. The sequence analysis was performed by automated Edman degradation of reduced and carboxymethylated insecticyanin and fragments generated therefrom by cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus proteinase. Most of the peptides were purified by reverse-phase high-performance liquid chromatography. A purification procedure for the isolation of insecticyanin in high yields and a simple method of determining disulfide linkages are also reported.

Amino Acid Sequence↗
PubMed1984

Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli.

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) inactivates dodecameric glutamine synthetase from Escherichia coli with concomitant labeling of one site/subunit (Foster, W.B., Griffith, M.J., and Kingdon, H.S. (1981) J. Biol. Chem. 246, 882-886). Cyanogen bromide cleavage of the FSBA-inactivated enzyme in 70% HCOOH produced a large peptide (Mr congruent to 4600) in approximately 75% yield containing N epsilon-4-carboxybenzenesulfonyl (CBS) lysine. The CBS-peptide was purified by high performance liquid chromatography on the basis of its relatively high UV absorbance at 246 nm (delta epsilon congruent to 19,000 M-1 cm-1). The appearance of the labeled peptide coincided with the loss of a peptide (lacking tyrosine and tryptophan) from the unmodified, active enzyme; the CBS-peptide also could be distinguished from cysteine-containing peptides labeled with thionitrobenzoate and from the adenylylated peptide. The identical peptide was labeled when the unadenylylated enzyme was inactivated with FSBA in the absence or presence of Mn2+ or Mg2+ (+/- L-glutamate) or when the enzyme was adenylylated. Conformational differences among these enzyme forms therefore are not detected by affinity labeling of ATP sites. The FSBA-labeled peptide spans residues 9-48 from the N-terminal end of the subunit polypeptide chain, and sequence analysis by automated Edman degradation revealed that CBS-lysine was at position 47. Thus, lysyl residue 47 appears to be near the subunit gamma-phosphate-binding site for ATP in the native glutamine synthetase structure. The primary structure of the CBS-labeled peptide is presented.

Adenosine↗
PubMed1984

Identification of the in vivo and in vitro phosphorylation sites of rat liver fructose 1,6-bisphosphatase.

Rat liver fructose 1,6-bisphosphatase appears to be unique in that it extends 24-26 residues beyond the COOH-terminal amino acid of other mammalian fructose 1,6-bisphosphatases and this extension contains phosphorylation sites. Using as a frame of reference the 335-residue sequence of pig kidney fructose 1,6-bisphosphatase (Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165), the rat liver enzyme would extend to residue 361. Limited proteolysis in the COOH-terminal region of the molecule with chymotrypsin, trypsin, or both sequentially, led us to establish that the phosphorylation sites are located at Ser residues 341 and 356. The in vitro phosphorylation of purified rat liver fructose 1,6-bisphosphatase by the catalytic subunit of cyclic AMP-dependent protein kinase results in modification at both residues, although the major site of phosphorylation (61%) is at Ser-341. In contrast, rat liver fructose 1,6-bisphosphatase purified from animals that had been injected with [32P] phosphate contains most of the label (81%) at Ser-356.

Amino Acid Sequence↗
PubMed1984

Identification of the highly reactive sulfhydryl group of pig kidney fructose 1,6-bisphosphatase at cysteine 128.

Fructose 1,6-bisphosphatases contain a highly reactive cysteine residue, the reactivity of which is influenced by ligands that bind at the catalytic and at the allosteric AMP sites of the enzyme. Nevertheless, the sulfhydryl group appears to be proximal to these sites and not a functional component of either. Modification of pig kidney fructose 1,6-bisphosphatase with three reagents, 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide, and phenacyl bromide, yields derivatives with similar properties, thus suggesting that the same residue was modified in each case. The modified enzymes exhibited: (a) higher Vmax when Mn2+ was used as the activating cation; (b) decreased activity in the presence of nonsaturating Mg2+ concentrations; (c) no change in sensitivity toward AMP inhibition. Automated Edman degradation of a tryptic peptide containing radioactive carboxamidomethylcysteine showed the sequence of residues Gly-111-Arg-140 of pig kidney fructose 1,6-bisphosphatase. The modified residue was shown to be cysteine-128, and the same cysteine residue was alkylated when the enzyme was reacted with phenacyl bromide. Cysteine-128 is also present in rat and sheep liver fructose 1,6-bisphosphatase and a long stretch of the sequence around this reactive cysteine residue is highly conserved.

Adenosine Monophosphate↗
PubMed1984

Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate.

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application.

Amino Acids↗
PubMed1984

Sequence of the bacteriophage SP01 gene coding for transcription factor 1, a viral homologue of the bacterial type II DNA-binding proteins.

The Bacillus subtilis phage SP01, whose DNA contains 5-hydroxymethyluracil (hmUra) in place of thymine, codes for an abundant, small, basic protein called TF1. TF1 binds preferentially to hydroxymethyluracil-containing DNA and thereby selectively inhibits transcription of such DNA in vitro. The gene for TF1 has been sequenced. We find that this viral protein is a homologue of the ubiquitous bacterial type II DNA-binding proteins. The three-dimensional structure of one of these bacterial proteins has recently been determined. We are able to discern common as well as distinctive features in the amino acid sequence and the three-dimensional structure of the homologous viral protein.

Amino Acid Sequence↗
PubMed1983

Amino acid sequence of the COOH-terminal region of fructose-1,6-bisphosphatases in relation to cyclic AMP-dependent phosphorylation.

Studies of in vitro phosphorylation of four different gluconeogenic fructose-1,6-bisphosphatases by the catalytic subunit of cyclic AMP-dependent protein kinase have shown that only rat liver fructose-1,6-bisphosphatase is a substrate of the protein kinase. A comparison of the molecular weights of fructose-1,6-bisphosphatases revealed that the nonphosphorylatable mouse liver, rabbit liver, and pig kidney enzymes have a subunit Mr approximately 37,000 while the subunit molecular weight of purified rat liver fructose-1,6-bisphosphatase is about 41,000 (Hosey, M. M., and Marcus, F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 91-94). To probe the structural basis for the higher molecular weight and unique phosphorylation of rat liver fructose-1,6-bisphosphatase, the CNBr fragment containing the phosphorylation site was purified and the amino acid sequence of this 43-residue peptide was determined. The sequence data revealed that the rat liver enzyme extends 24-26 residues beyond the COOH-terminal amino acid of pig kidney and rabbit liver fructose-1,6-bisphosphatase and that cyclic AMP-dependent phosphorylation sites are located in this proline-rich extension. The kinetic properties of rat liver fructose-1,6-bisphosphatase do not appear to be influenced in any way, either by the COOH-terminal extension itself or by the state of phosphorylation. Polyacrylamide gel electrophoresis of immunoprecipitates from crude extract supernatants demonstrated that the rat liver enzyme is larger than other fructose-1,6-bisphosphatases studied to date, and that the differences in molecular weight are not due to proteolytic modification of other fructose-1,6-bisphosphatases during isolation procedures.

Amino Acid Sequence↗
PubMed1982

The covalent structure of apolipoprotein A-I from canine high density lipoproteins.

The complete amino acid sequence of apolipoprotein A-I (apo-A-I) from canine serum high density lipoproteins (HLD) has been determined by automated Edman degradation of the intact protein and proteolytic fragments derived therefrom. The major strategy involved analysis of overlapping sets of peptides generated by cleavage at lysyl residues with Myxobacter protease and by tryptic hydrolysis at arginines in the citraconylated protein derivative. Canine apo-A-I has 232 residues in its single polypeptide chain and its covalent structure is highly homologous to one of the two reported sequences for human apo-A-I. As in the case for the human apoprotein, predictive analysis of the canine apo-A-I sequence suggests that it comprises a series of amphiphilic alpha helices punctuated by a periodic array of prolyl residues. Human HDL contains a second major protein component, apolipoprotein A-II (apo-A-II) that is lacking in HDL from dog serum. The absence of apo-A-II in canine HDL raised the possibility that the apo-A-I from this source might contain within its primary structure sequences related to apo-A-II and thus perform the dual function of both proteins in one. Our analysis proves that canine apo-A-I has all of the structural features of human apo-A-I and that it is not an A-I: A-II hybrid molecule.

Amino Acid Sequence↗
PubMed1982

Complete amino acid sequence of pig kidney fructose-1,6-bisphosphatase.

The covalent structure of the pig kidney fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) subunit has been determined. Placement of the 335 amino acid residues in the polypeptide chain was based largely on automated Edman degradation of eight purified cyanogen bromide fragments generated from the S-carboxymethylated protein. The determination of the amino acid sequence of the largest cyanogen bromide fragment (154 residues) required additional analysis of subfragments obtained by tryptic cleavage at arginyl residues and by mild acid cleavage of an Asp-Pro peptide bond. Alignment of the cyanogen bromide fragments was accomplished by analysis of a product of limited proteolysis by an endogenous protease and by characterization of the tryptic peptides isolated from S-[14C]carboxymethylated fructose-1,6-bisphosphatase. This sequence information has permitted the identification of several reactive sites of functional and structural significance in pig kidney fructose-1,6-bisphosphatase.

Amino Acid Sequence↗
PubMed1981

Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120.

A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described. The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits. Sequence analysis suggests that the subunits are identical. There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli. NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources. The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E. coli.

Amino Acid Sequence↗
PubMed1981

The covalent structure of pig kidney fructose-1,6-bisphosphatase. Sequence of a 63-residue cyanogen bromide peptide containing a phosphorylatable serine.

Native pig kidney fructose-1,6-bisphosphatase, in contrast to the rat liver enzyme, is not a substrate of cyclic AMP-dependent protein kinase. However, the pig kidney enzyme becomes a substrate when phosphorylation is performed in 1.6 M urea, after prior unfolding in 8 M urea. A cyanogen bromide fragment containing the phosphorylation site has been isolated and the amino acid sequence of this 63-residue peptide has been determined. This peptide has the following sequence: Leu-Asp-Pro-Ala-Ile-Gly-Glu-Phe-Ile-Leu-Val-Asp-Arg-Asn-Val-Lys-le-Lys-Lys-Lys- Gly-Ser(P)-Ile-Tyr-Ser-Ile-Asn-Glu-Gly-Tyr-Ala-Lys-Glu-Phe-Asp-Pro-Ala-Ile-Thr- Glu-Tyr-Ile-Glu-Arg-Lys-Lys-Phe-Pro-Pro-Asp-Asn-Ser-Ala-Pro-Tyr-Gly-Ala-Arg-Tyr -Val-Gly-Ser-Met. The amino acid sequence around the phosphorylated serine residue resembles those of other protein substrates of cyclic AMP-dependent protein kinase, but it is completely different from the phosphorylation site found in native rat liver fructose-1,6-bisphosphatase.

Amino Acid Sequence↗
PubMed1981

Structure and action of heteronemertine polypeptide toxins. Amino acid sequence of Cerebratulus lacteus toxin B-II and revised structure of toxin B-IV.

The primary structure of Cerebratulus lacteus toxin B-II has been investigated by automated Edman degradation of the reduced, carboxymethylated protein and of tryptic peptides derived from the maleylated protein. As a result of these studies, the identity of all 55 amino acid residues in the polypeptide chain has been unambiguously determined. Hydroxyproline, an amino acid not normally found in nonfibrous proteins, occupies position 10 of toxin B-II. The sequence analysis of B-II led us to re-examine the sequence of Cerebratulus toxin B-IV. The revised structure of toxin B-IV is presented herein. Like B-II, toxin B-IV contains hydroxyproline at position 10 of its sequence. Comparison of the sequences of toxins B-II and B-IV reveals a high degree of homology between these two polypeptides, particularly within the NH2-terminal two-thirds of each chain.

Amino Acid Sequence↗
PubMed1981

Studies on the phosphorylation of muscle phosphofructokinase.

Phosphorylation of rabbit skeletal muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase occurs with a Km of about 230 microM and Vmax approaching that seen with histone as a substrate. The rate of phosphorylation of phosphofructokinase by protein kinase is increased by allosteric activators of phosphofructokinase, whereas inhibitors of phosphofructokinase inhibit the phosphorylation. Inhibitors and activators change Vmax but not Km. The site of phosphorylation is a serine residue that is the sixth amino acid from the carboxyl terminus. Limited proteolysis by trypsin releases an octapeptide from the carboxyl terminus and a brief exposure to subtilisin releases a dodecapeptide from the carboxyl end. The sequence of the dodecapeptide is His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu-Ala-Thr-Val. Phosphofructokinase isolated from a rabbit injected 18 h prior to killing with [32P]PO4 contained covalently bound radioactive phosphate. Approximately 80% of the phosphate was released in a trichloroacetic acid-soluble form following limited proteolysis by trypsin, under which conditions the enzyme remained with a monomer size of about 80,000 daltons. The position of elution from Sephadex G-25 of the phosphopeptide was identical with that found following limited trypsin proteolysis of in vitro labeled enzyme. Migration of the phosphopeptides on thin layer cellulose chromatography was also identical. We conclude that at least 80% of the radioactive phosphate introduced within 18 h of an intravenous injection of [32P]PO4 is found at the same site as that introduced by phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase.

Amino Acid Sequence↗