Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A1

Renatured, S-carboxymethylated subunit A₁ of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, $\text{102}$, 1021). In the absence of acceptor self ADP-ribosylation of A₁ subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO₄(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A₁ was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A₁ subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg¹-Asp-Arg-Tyr. The Arg¹ in the peptide was indicated to be the site of ADP-ribosylation.     

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b₁, -b₂, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b₁, -b₂, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A₂. The cytochrome photooxidation and P430 and A₂ photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A₂ was least active in the cytochrome photooxidation. A₁, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes (Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys.219, 209–218). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A₁, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A₂. The 13,000-dalton polypeptide which is associated with CP1-a, -b₁, and -b₂ is not required for the functioning of the reaction center.     

Complete primary structure of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.     

Continuous fluorometric assay of phospholipase A2 with pyrene-labeled lecithin as a substrate

1,2-Bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine was synthesized as a fluorogenic substrate for phospholipase A₂. It has a critical micellar concentration of 7.3 μm and gives only excimer fluorescent emission at 480 nm in aqueous micellar dispersion. When hydrolyzed by phospholipase A₂, the products give only monomer emission which is monitored best at 382 and 400 nm. Conditions were developed for an assay for phospholipase A₂ using this substrate. The assay was sensitive to as little as 8 ng of pure porcine pancreatic phospholipase A₂.     

Molecular conformation of prostaglandin A1 monoclinic cyrstalline polymorph

The molecular conformation of the monoclinic crystalline polymorph of prostaglandin A₁ has been determined by X-ray diffraction techniques. The space group is P2₁ with a = 13.637 (2), b = 7.567 (1), I c = 10.576 (2) Å, β = 107.37 (3)°; D_c = 1.073 g·cm⁻³ for Z = 2. The molecular conformation is characterized by the nearly parallel arrangement of the C₁–C₇ and C₁₃–C₂₀ side chains, with a general flattening of the overall structure when compared with the orthorhombic polymorph. The cyclopentenone moiety assumes a C₈ envelope conformation with C₈ and O₉ displaced +0.29 Å and −0.18 Å from the C₉–C₁₀=C₁₁–C₁₂ plane respectively. Concerted, small variations of the torsion angles, primarily about the C₈–C₁₂, C₁₄–C₁₅ and C₁₆–C₁₇ bonds, bring the monoclinic and orthorhombic conformations into coincidence.     

Structure and synthesis of malformin A1

The structure of malformin A₁, a metabolic product of Aspergillus niger, was reexamined and the sequence of its amino acid constituents established as

The cyclopentapeptide-disulfide corresponding to this structure was prepared through stepwise synthesis of the protected pentapeptide derivative, benzyloxy-carbonyl-l-isoleucyl-S-benzyl-d-cysteinyl-S-benzyl-d-cysteinyl-l-valyl-d-leucine methylester, which in turn was converted to the hydrazide, partially deprotected, and cyclized via the azide. On removal of the S-benzyl groups and oxidation to the disulfide, a synthetic material was obtained that was indistinguishable from natural malformin A₁ and was as equally potent in causing curvatures on corn roots.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

Modification of chloroplast CF1 by fluoresceinisothiocyanate

Incubation of the chloroplast coupling factor with fluorescein isothiocyanate inhibits the Ca-ATPase activity of the enzyme and results in incorporation of fluorescein into the α and β subunits. High concentrations of ATP prevent the inhibition and reduce the incorporation of fluorescein into the α and β subunits. Ca and Mg ions increase fluorescein isothiocyanate inhibition and fluorescein incorporation into the α, β and γ subunits. It is suggested that fluorescein isothiocyanate modifies the catalytic site of the enzyme by blocking lysine residues in the α and β subunits which are involved in the binding of ATP.     

Influence of the estrous cycle on heterosexual aggression in two strains of rats (S3 and WEzob)

Two strains of rats (S₃ and WEzob), which show different levels of aggression in the laboratory, were tested in repeated heterosexual confrontations. Daily 15-min observations were made of the interactions between a female throughout a complete estrous cycle and the same male partner. In both strains the topography of aggression was similar in males and females, but the frequency of specific parameters varied. Males showed more offensive and females more defensive patterns. The overall level of aggression was very low on the day of estrous, when the female was sexually receptive. There were no differences in any elements of female or male behavior between the other 3 days of the cycle. The results support previous conclusions from single-sex encounters that in rats there is no sexual dimorphism in the ability to show aggression.     

Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vitamin K₁ hydroquinone has been identified as a metabolite of vitamin K₁ biotransformation catalyzed by highly purified DT-diaphorase (NAD(P)H dehydrogenase, EC 1.6.99.2) isolated from livers of 3-methylcholanthrene induced rats. The hydroquinone was sufficiently stable to permit enzymatic reactions to be conducted under an atmosphere of air and quantitation of hydroquinone by high performance liquid chromatography. Based on kinetic data reported here, warfarin and probably dicoumarol at therapeutic levels do not appreciably affect DT-diaphorase catalyzed vitamin K hydroquinone formation.     

The primary structure of ribosomal protein S7 from E. coli strains K and B.

Ribosomal proteins S7 from 30S subunits of Escherichia coli strains K and B differ extensively in their aminoacid compositions. The experimental details which led to the determination of the complete primary structures of proteins S7K and S7B are presented. Protein S7K consists of a single polypeptide chain of 177 aminoacids giving a calculated molecular weight of 19, 732, whereas protein S7B has 153 residues which amount to a molecular weight of 17,131. Aminoacid sequences were determined by a combination of automated Edman degradation of the intact proteins in a modified Beckman sequenator and sequencing of peptides obtained by digestion with trypsin. Staphylococcus aureus protease, thermolysin and pepsin, either by solid-phase Edman degradation or by dansyl-Edman degradation. Additional information about the primary structure was derived from peptides resulting from chemical cleavages of the protein by 2-(2-nitrophenyl-sulphenyl)-3-methyl 3' bromoindolenine at its tryptophanyl bonds and by cyanogen bromide at its methionyl bonds leading to large fragments. The mutational event occurring between S7B and S7K was characterized. Protein S7K contains an additional sequence of 24 aminoacids at its C-terminal end. The aminoacid sequence of both proteins S7K and S7B was compared to the published sequences of the other ribosomal proteins of Escherichia coli and predictions for the secondary structure of these proteins were made.     

Analysis of catalytic subunit microheterogeneity in ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum

Urea isoelectric focusing of dissociated, carboxymethylated Nicotiana tabacum ribulose-1,5-bisphosphate carboxylase/oxygenase reveals catalytic subunit microheterogeneity. Aggregated or nonaggregated sucrose gradient-purified preparations and the crystalline protein displayed essentially identical large subunit multiple polypeptide patterns. Various pretreatments which fully dissociate the holoenzyme did not alter catalytic subunit microheterogeneity. Direct comparison of the carboxymethylated and noncarboxymethylated crystalline and sucrose gradient-purified proteins demonstrated that the large subunit multiple polypeptide pattern was not an artifact of carboxymethylation. The inclusion of the seryl protease inhibitor phenylmethylsulfonyl fluoride during purification of the holoenzyme did not affect the large subunit multiplicity. However, the addition of leupeptin, a potent thiol proteinase inhibitor, to all solutions during purification of the native protein markedly reduced large subunit polypeptide L₃ and increased the staining of polypeptide L₂, suggesting that L₃ is a leupeptin-sensitive proteinase degradation product of L₂. Polypeptide L₁ also appeared to be a purification-related artifact, but derived from a modification of L₂ other than that which yielded L₃. We conclude that polypeptide L₂ is the single, native isoelectric form of the catalytic subunit of tobacco ribulosebisphosphate carboxylase/oxygenase.