Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

The activation of bovine Factor X by bovine Factor Xa

A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor X_a, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca²⁺ showing the greatest stimulatory effect and Mn²⁺ exhibiting a lower degree of activity for this reaction. Although Sr²⁺ and Mg²⁺ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca²⁺. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-X_a, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor X_a (Factor β-X_a were produced. At saturating levels of Ca²⁺, the K_{m app} for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min^?1 mol^?1 Factor X_a. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min^?1 mol^?1 Factor X_a.     

Non-random layout of the amino acid loci on the genome of Escherichia coli

A strong correlation exists between the relative frequencies of occurrence of the amino acids in bulk Escherichia coli protein and their genetic map positions when the latter are indexed against the position of the origin of DNA synthesis. The greater the production of the amino acid, the closer its operon is to the origin.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

Messenger RNAs from the transforming region of bovine papilloma virus type I

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.     

Is vitamin E the only lipid-soluble,chain-breaking antioxidant in human blood plasma and erythrocyte membranes?

The concentration of lipid-soluble, chain-breaking antioxidants in human plasma and in erythrocyte ghosts have been determined for the first time by an inhibited-autoxidation method. The results are very similar to the concentrations of vitamin E measured for the same blood components by the HPLC method. It is concluded that vitamin E, which is largely present as alpha-tocopherol, is the only significant lipid-soluble, chain-breaking type of antioxidant present in human blood. The concentration of vitamin E in the plasma lipids divided by the concentration of vitamin E in the ghost membrane lipids is approximately a constant despite the large differences in vitamin E-intake and in plasma lipid concentrations in different individuals. Vitamin E/lipid ratios for plasma and ghosts were larger for subjects taking a supplement of alpha-tocopherol acetate of 100 IU per week, compared to nonsupplemented subjects (based on data from a limited number of subjects). A larger supplement of 2800 IU per week did not significantly increase the vitamin E/lipid ratios.     

Crystallographic data on a complete kappa-type human Bence-Jones protein.

A complete human κ-type Bence—Jones protein (Fin) has been isolated and crystallized. Immunochemical and physicochemical characterization of protein Fin indicates that it is of the κ-chain subgroup, κII, and that it consists of two non-covalently bound intact monomers having a molecular weight of ~23,000 Crystals of Bence—Jones protein Fin obtained from ammonium sulfate solutions have the orthorhombic space group P2₁2₁2₁ with cell dimensions $\text{a = 132.0}\text{A}\text{?}\text{, b = 93.3}\text{A}\text{?}\text{, and}\text{c = 42.3}\text{A}\text{?}$. The asymmetric unit consists of a dimer of molecular weight ~46,000.     

A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain

Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D_{20, W} = 3.1 × 10^?7 cm² s^?1 and the value of D_{20, W} = 5.1 × 10^?7 cm² s^?1 calculated for tubulin dimer of M_rel 100,000. The 30 S ring has an observed diffusion coefficient of D_{20, W} = 0.49 × 10^?7 cm² s^?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.     

Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli

The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.     

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10^?4m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC^? lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC^? enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC^? adenylate cyclase utilized MgATP^2? as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC^? enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP^2?. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.     

Demetallation and esterification reactions of heterocyclic amino acid complexes

The copper(II) complex of 4-methyloxazolidine- 4′-carboxylic acid and the nickel(II) complex of 3N,7N-(1,3,5,7-tetraazabicyclo [3.3.1]nonyl)diacetic acid were prepared and then treated with sodium borohydride to form the sodium salts of the respective acids. The saturated heterocyclic rings of the acids are retained in the reactions.The methyl esters of the acids were subsequently prepared under anhydrous conditions and characterized by gas chromatography/mass spectrometry (GC/MS).     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

The effects of mepacrine and p-bromophenacyl bromide on arachidonic acid release in human platelets

Two inhibitors of thrombin-stimulated arachidonic acid release from platelets, p-bromophenacyl bromide and mepacrine, were examined for their ability to inhibit the phospholipase C-diglyceride lipase pathway. This pathway involves hydrolysis of phosphatidylinositol to diglyceride, followed by release of arachidonate from diglyceride, and has been proposed as an alternative or addition to phospholipase A₂ as a mechanism for arachidonate release. p-Bromophenacyl bromide, a potent alkylating agent, was shown to cause a time-dependent inhibition of phosphatidylinositol-specific phospholipase C activity in crude platelet extracts; the inhibition was >90% after 15 min incubation with 100 μmp-bromophenacyl bromide. However, p-bromophenacyl bromide was also shown to destroy about one-half of the titratable sulfhydryl groups in whole platelets under similar conditions. The lack of specificity of p-bromophenacyl bromide was further demonstrated by our finding that thrombin-stimulated serotonin release was also inhibited by conditions inhibiting arachidonate release and that diglyceride lipase activity was decreased by higher levels of p-bromophenacyl bromide. Mepacrine was found to inhibit the activity of phosphatidylinositol-specific phospholipase C and had a greater effect at low substrate concentrations. The loss of [¹⁴C]arachidonate from both endogenous phosphatidylinositol and phosphatidylcholine in intact platelets was also inhibited. Thrombin-stimulated serotonin release was impaired by mepacrine also but only at a concentration 10-fold greater than that required to prevent arachidonate release. Thus we have shown that these two agents which inhibit arachidonate release are inhibitors of the phosphatidylinositol-specific phospholipase C-diglyceride lipase pathway. The multiple effects produced by both compounds limit their utility as agents to examine the source and mechanism of arachidonate release.     

A calorimetric comparison of structural transitions of erythrocyte ghosts from normal individuals and from patients with muscular dystrophy

Using a highly sensitive scanning calorimeter, the thermally induced structural transitions of erythrocyte ghosts from normal individuals and from patients with Duchenne muscular dystrophy (DMD) were carefully examined. No differences were observed under a variety of conditions. This finding is consistent with the idea that the composition, structure, and organization of membrane proteins and lipids in DMD erythrocyte membranes is very similar to normal erythrocyte membranes, in contrast to many other reports in the literature which utilized different techniques.     

Metal complexes of N-hydroxy-imino-di-α-propionic acid and related ligands

N-hydroxy-imino-di-α-propionic acid, the ligand present in the natural oxovanadium(IV) complex ‘amavadin’ which occurs in the toadstool Amanita muscaria, has been synthesised, as well as two related ligands—N-hydroxy-iminodiacetic acid and imino-di-α-propionic acid—useful for comparison purposes. The formation of complexes of these ligands with VO²⁺, Ni²⁺ has been studied and their stability constants have been determined.The two N-hydroxy-substituted ligands, of low basicity, form ML₂ complexes with VO²⁺, unlike the more basic derivatives of iminodiacetic acid. Since substitution of ligands bonded to the apical site trans to the oxo ligand is very fast and the formation of ML₂ complexes of VO²⁺ exposes that apical site to the reaction media, this may be the reason why oxovanadium(IV) and the unusual derivative of iminodiacetic acid present in ‘amavadin’ were selected for the biological role that this complex plays in the toadstool.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

Conservation and DNA sequence arrangement of the DNA polymerase I gene region from Klebsiella aerogenes, Klebsiella pneumoniae and Escherichia coli

Linked multiple mutation is observed after treatment of Escherichia coli with methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and N-ethyl-N-nitro-N-nitrosoguanidine but not ultraviolet light. Induction of linked multiple mutations requires the uvrE⁺ gene product indicating the involvement of the mismatch repair system. The observation of linked multiple mutations is not due to mutagenesis occurring in a subpopulation of cells. Growing point mutagenesis also occurs after treatment with these mutagens but not with ultraviolet light. It is likely that the excess of mutations observed with these mutagens at growing points is at least partly a relative effect, rather than one due to an absolute increase of reactivity at the DNA growing point region. This relative effect may result from the operation of an inducible repair mechanism which removes O⁶-alkylguanine residues from the DNA distal to the bacterial growing point. The adaptive response, first described by Robins &; Cairns (1979) prefers O⁶-methylguanine over O⁶-ethylguanine.     

An investigation by ion-exchange chromatography of a chromium,amino acid and nicotinic acid mixture

The mixture of chromium, nicotinic acid and the amino acids glycine, glutamic acid and cysteine which stimulates the rate of CO₂ production in a yeast bioassay system was subjected to the separation scheme based on ion-exchange chromatography which has been used to separate the chromium- containing fractions in brewer's yeast, [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., 18, 195 (1983)]. Four chromium-containing fractions (C2 to C5) were obtained by salt gradients and two further fractions (G1 and G2) were obtained using a pH gradient. All were amino acid-containing complexes of chromium and all except C5 also contained nicotinic acid. However, none of the isolated chromium fractions showed any activity in a yeast bioassay. On the basis of previous work, the activity of the original mixture was attributed to the presence of an oxygen-coordinated trans chromium(III)-dinicotinate complex. Biologically- inactive chromium complexes such as Cr(glu)₂(H₂O)⁺₂ and Cr(gly)₂(H₂O)⁺₂ after elution by ammonium hydroxide from Dowex 50W-X12 cation- exchange columns, stimulated the rate of CO₂ production in the yeast bioassay. Elution with other bases, such as lithium hydroxide, potassium hydroxide and sodium hydroxide led to inactive fractions in all cases. A warning is therefore given that the use of ammonium hydroxide-elution of ion-exchange columns to isolate glucose tolerance factor fractions from biological samples (such as brewer's yeast) can lead to active fractions which do not relate to the native material.