Studies on the structure of rabbit muscle aldolase: Ordering of the tryptic peptides; Sequence of 164 amino acid residues in the NH2-terminal BrCN peptide

The sequence of 164 amino acid residues in the NH₂-terminal BrCN peptide of rabbit muscle aldolase has been determined. The information has permitted location of the following amino acid residues involved in the catalytic activity or in maintaining the structural integrity of the enzyme: Cys-72, forms a disulfide bridge with Cys-336 in the COOH-terminal segment on inactivation of the enzyme by oxidation; Lys-107, forms a Schiff base with pyridoxal phosphate upon inactivation of aldolase by this reagent; Cys-134 and Cys-177, buried, do not react with SH-reagents in the native enzyme.     

The cyanogen bromide peptides of the apoprotein of low density lipoprotein (ApoB): its molecular weight from a chemical view.

After >95% cleavage of the apoprotein (apoB) of the low density lipoproteins with cyanogen bromide, the peptides produced are shown to be extensively aggregated in sodium dodecyl sulfate. Both high temperature and increased concentration (5%) of the detergent are necessary to shift the aggregated peptides from high molecular weight (>25,000) to lower molecular weight aggregates as seen on sodium dodecyl sulfate polyacrylamide gel electrophoresis. End group analyses of the cyanogen bromide digestion by automated sequencer techniques indicate the presence of five (5) methionines. With a known methionine content of 16 moles/100,000 g protein, the molecular weight of the apoprotein must be approximately 30,000.     

Recovery and analysis of peptides from thin layer cellulose

Methods are described which permit the recovery and analysis of peptides at the nanomole level in quantitative yield from cellulose thin layers. Although commercial celluloses are contaminated with significant levels of proteins which are not removed by washing and which interfere with amino acid analyses at the nanomole level, the methods described avoid these contaminants and permit amino acid analyses on the extracted peptides. The effect of reaction with ninhydrin or fluorescamine on the recovery of peptides is also described.     

Vasoactive intestinal peptide (VIP) from chicken: Synthesis and properties of the C-terminal hendecapeptide

The hendecapeptide, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Thr-NH₂, corresponding to sequence 18–28 of chicken vasoactive intestinal peptide (VIP), was synthesized stepwise, starting with the C-terminal residue. The in situ technique was applied; o-nitrophenyl esters and p-nitrophenyl esters were used for acylation. The product was compared with, and found indistinguishable from, the C-terminal cyanogen bromide fragment of natural chicken-VIP. Some pharmacological properties of the hendecapeptide were also determined. In two separate experiments, the chain of the hendecapeptide was further lengthened to encompass residues 14–28 of chicken-VIP but with leucine and norleucine in place of methionine in position 17. The two pentadecapeptides showed biological activities comparable to those of the C-terminal pentadecapeptide fragment of porcine VIP or its 17-norleucine analog.     

First observation of a beta-turn conformation fused with the oxy-analogue of an alpha-turn: the molecular structure of a model peptide of the C-terminal part of gramicidin A

The molecular structure of N-tert-butyloxycarbonyl-D-leucyl-L-phenylalanyl ethanolamide (t-Boc-D-Leu-L-Phe-EA), a protected analogue of the C-terminal dipeptide of the membrane-active linear antibiotic gramicidin A, has been determined by X-ray diffraction. One of the two independent molecules in the asymmetric unit is characterized by a chain reversal stabilized by an intramolecular, three-centre, double hydrogen bonding. It represents the first experimental evidence for a beta-turn conformation fused with the oxy-analogue of an alpha-turn.     

Variations in the amounts of RNA polymerase forms I, II and III during preimplantation development in the mouse.

DNA-dependent RNA polymerase has been measured at various stages of preimplantation development in mouse embryos. The total RNA polymerase activity per embryo increases rapidly from the 8-cell stage to the blastocyst stage. Studies with low α-amanitin concentrations, which inhibit form II RNA polymerase, and high α-amanitin concentrations, which inhibit both form II and III RNA polymerases indicate that the relative proportions of the three forms change significantly during preimplantation development. The changes which occur in the types and levels of RNA polymerase appear to parallel corresponding changes in the synthesis of the major classes of RNA.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.     

Dynamic information from protein crystallography. An analysis of temperature factors from refinement of the hen egg-white lysozyme structure.

A preliminary analysis is presented of whether the isotropic temperature factors derived from refinement of the tetragonal crystal structure of hen egg-white lysozyme at 2 Å resolution can be interpreted in terms of molecular motion. If the contributions to the temperature factors from experimental errors, crystal disorder and imperfections of the molecular model are neglected, the apparent thermal motion is found to be compatible with the pair of molecules that have the strongest interactions in the crystal either moving as a rigid-body with libration about a common axis, or vibrating in an intramolecular mode with the principal amplitudes radial to this axis. There is only weak evidence for hingebending, i.e. that the two lobes of lysozyme vibrate so that their separation varies. Side-chains that are exposed to the solvent and some segments of the main polypeptide chain (including residues 70–73, 82–84, 101–103) have greater apparent thermal motion than the remainder of the molecule.Although this analysis at a single temperature cannot establish whether thermal motion or static disorder (including conformational variability) underlies the observed effects, it suggests that the accurate determination of temperature factors will be useful in detailed studies of the dynamic properties of macromolecules.     

Simultaneous analysis of pipecolic acid with proline in the brain by selected ion-monitoring technique

A method for the simultaneous analysis of pipecolic acid and proline in the brain is developed. The qualification and quantification of pipecolic acid and proline are accomplished with gas chromatography/mass spectrometry including a selected ion-monitoring technique by using deuterium-labeled proline as an internal standard, after the amino and carboxylic groups of these cyclic amino acids are derivatized with boron trifluoride methanol complex and heptafluorobutyric anhydride. The lower limit of quantification for the method is picomole levels and the concentration of pipecolic acid and proline in rat whole brain is determined to be 1.05 and 71.50 nmol/g of tissue, respectively.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

The isolation of a peptidyl dipeptidase from mouse brain cytosol that cleaves adrenocorticotropic hormone-(7-10) and des-tyrosine-enkephalins

An enzyme present in mouse brain cytosol cleaves C-terminal dipeptides from substrates including ACTH-(7-10) (Phe-Arg-Trp-Gly), and des-Tyr-[Met]- and des-Tyr-[Leu]enkephalin. By means of ion-exchange chromatography and gel filtration, the peptidase was purified to a specific activity of 1570 times that of brain homogenate. At this purification, a second peptidase, which hydrolyzes Trp-Gly and other peptides [M. E. A. Reith and A. Neidle (1979) Biochem. Biophys. Res. Commun. 90, 794-800] was still present, but could be removed by preparative polyacrylamide gel electrophoresis. The des Tyr-enkephalin-cleaving enzyme has a molecular weight of about 85,000 and a pH optimum of 7.8. It is inhibited by metal-chelating and sulfhydryl reagents. The enzyme has a strong preference for substrates with an aromatic residue in the position adjacent to the C-terminal amino acid, although some peptides meeting this criterion were competitive inhibitors rather than substrates. Peptides with less than four residues were inactive and, in general, tetrapeptides were found to be more reactive than larger analogs, when peptides with common C-terminal sequences were compared. The peptidyl dipeptidase, which has not been described previously, can be readily distinguished from angiotensin-converting enzyme (EC 3.4.15.1) and from neutral endopeptidase (EC 3.4.24.11) by its subcellular localization, substrate specificity, and response to inhibitors. It was suggested that peptidyl dipeptidase-B (PDP-B, EC 3.4.15.-) would be an appropriate name for the enzyme. PDP-B is widely distributed among mouse tissues.     

Crystal and molecular structure of [Cu(5'-inosine monophosphate) (dipyridylamine) (H2O)]2.4H2O.

The ternary complex [Cu(5′-IMP)(dpa)(H₂O)]₂ has been prepared and its structure analyzed by x-ray diffraction. It has a dimeric structure in which the 5′-IMP ligands coordinate solely through their phosphate groups. This geometry is in marked contrast to that of another $\text{Cu}\text{5′-}\text{IMP}$ ternary complex, [Cu(5′-IMPH)(bipy)(H₂O)₂]⁺, which shows metal binding through the purine base rather than the phosphate group.     

An improved method for the separation of peptides and alpha-amino acids on copper-Sephadex.

An improved method for the separation of peptides from large amounts of α-amino acids on copper-Sephadex is described. The separation is essentially dependent upon the copper content of Sephadex, the pH of the system, and the concentration of the sample, and it is due to the different stabilities of copper complexes of Sephadex, peptides, and α-amino acids. Sephadex behaves as a solid ligand. Compounds that form weaker complexes with copper than Sephadex apparently move with the solvent front. α-Amino acids that form copper complexes having a stability comparable to that of copper-Sephadex complexes are retained on the column. Peptides form strong complexes, stripping copper from the column. They are only slightly retained. The method is most practical when copper is removed from the eluates with chelating ion-exchanger Dowex A-1. Examples are given for the chromatography of single compounds, model mixtures, and extracts from cheese and yeast.     

Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region.

The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 10⁶. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T₁-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins.     

Effect of atrial natriuretic factor (ANF)-related peptides on aldosterone secretion by adrenal glomerulosa cells: critical role of the intramolecular disulphide bond

We previously demonstrated that synthetic 48-73 atrial natriuretic factor (ANF) (previously called 8-33 ANF) blocked the response of rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. We have now investigated the effects of natural 43-73 ANF, oxidised synthetic 48-73 ANF and the natural 1-73 ANF on aldosterone output by rat glomerulosa cells. The natural 43-73 ANF and the natural 1-73 ANF were equipotent to 48-73 ANF in inhibiting the stimulation of aldosterone secretion produced by angiotensin II with an IC50 of 2 X 10(-9)M. Similar results were obtained with ACTH and potassium. After oxidation with performic acid, 48-73 ANF was completely devoid of activity on the response of aldosterone to angiotensin II, ACTH and potassium. We conclude that the intramolecular disulphide bond in 48-73 ANF is critical for maintaining the active conformation of ANF.     

Studies of adenosine triphosphate transphosphorylases. XIV. Equilibrium binding properties of the crystalline rabbit and calf muscle ATP--AMP transphosphorylase (adenylate kinase) and derived peptide fragments.

Both a fluorescence-quenching technique and a uv-difference spectral method have been used to study the binding of 1,N⁶-etheno analogs of the adenine nucleotides (?ATP, ?ADP, ?AMP) (J. A. Secrist III, J. R. Barrio, N. J., Leonard, and G. Weber, 1972, Biochemistry, 11, 3499–3506) to crystalline rabbit and calf muscle ATP-AMP transphosphorylase in the presence and absence of Mg²⁺, at 0.16 ($\text{Γ}\text{2}$), 25 °C, and pH 7.4. In addition, the binding of the ?-analogs of the adenine nucleotides has been studied to two S-[¹⁴C]carboxymethylated peptide fragments of the rabbit muscle enzyme (residues 1–44 = MT-I; residues 171–193 = MT-XII), as well as to a synthetic nonapeptide corresponding to residues 32 ? 40 of the rabbit muscle enzyme. In the case of the rabbit and calf enzymes: Mg?ATP^2?, ?ATP^4?, Mg?ADP^?, and ?AMP^2? are bound stoichiometrically (n ～- 1), Mg?AMP is insignificantly bound, and n ～- 2 for ?ADP^3? (n = maximal number of moles bound per mole of protein). In the case of S-carboxymethylated peptide fragments: MT-I binds stoichiometrically to Mg?ATP^2?, ?ATP^4?, Mg?ADP^?, and ?ADP^3? with values of n ～- 1; but MT-I does not bind to ?AMP^2? significantly. MT-XII binds stoichiometrically to uncomplexed ?AMP^2? or to uncomplexed ?ADP^3? (both with n ～- 1); whereas, the binding of Mg?ADP^?, ?ATP^4?, and Mg?AMP to MT-XII are comparatively insignificant. Other peptide fragments in the molecule, viz. fragments MT-IV (residues 77–96) or MT-VI (residues 106–126) did not bind significantly to any of the ethenoanalogs; nor did insulin, nor, e.g., did bo vine serum albumin. The binding of the etheno analogs was also studied to an equimolar mixture of peptides MT-I + MT-XII, which qualitatively duplicated the binding pattern of the entire native molecule, and except for ?ATP^4? or Mg?ATP^2? (which are bound more tightly to the entire native molecule), even quantitatively. The synthetic peptide (residues 32 to 40) was found to bind to Mg?ATP^2?, ?ATP^4?, and Mg?ADP^?, with n ～- 1; but it does not significantly bind to ?AMP^2?, nor to ?ADP^3?. These binding data support the idea that there are two separate sites for the binding of either (a) the complexed nucleotide substrate (MgATP^2? or MgADP^?) residing in the sequence of MT-I (residues 1 to 44) and in the neighborhood of residues 32 to 40, or (b) the uncomplexed nucleotide substrate (AMP^2? or ADP^3?) residing in the sequence of MT-XII (residues 171 to 193) of the rabbit muscle enzyme.     

Identification of a peptide sequence involved in association of subunits of yeast hexokinases

The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver

The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm² with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA.