The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high $\text{pH}\text{6.8}\text{pH}\text{8.2}$ activity ratio (0.4 – 0.7) and a high level of Ca²⁺ independent activity $\text{(EGTA}\text{Ca}{}^{\mathrm{2+}}\text{= 0.3?0.5)}$. The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a M_r 128,000 polypeptide in the gradients. This polypeptide and a M_r 143,000 polypeptide were labeled with ³²P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a M_r 102,000 membrane polypeptide which followed the distribution of Ca²⁺-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A M_r 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca²⁺/calmodulin-stimulated manner, including a M_r ca. 72,000 polypeptide found only in the transverse tubule.     

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ($\text{w}\text{v}$) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.     

Phosphoprotein-phosphatase activity associated with human placental alkaline phosphatase.

Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein phosphatase activity. This was demonstrated by the dephosphorylation of ³²P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase $\text{a}$ phosphatase activity.     

Unit cell transormations in yeast phenylalanine transfer RNA crystals

The orthorhombic unit cell of crystalline yeast phenylalanine transfer RNA has dimensions $\text{a = 33}\text{A}\text{?}$, $\text{b = 56}\text{A}\text{?}$ and $\text{c = 161}\text{A}\text{?}$. When the mother liquor dries partially, a series of transformations takes place in which the a and b axes change very little but the c axis decreases abruptly first to 128 Å and then to 109 Å. In a closely related orthorhombic cell in a different space group the c axis is 104 Å. Although there is some loss in resolution in these smaller unit cells, the over-all distribution of scattering intensity does not change substantially. This suggests that the tRNA molecules can slide together along the c axis without a substantial change in internal structure.     

Regulation of muscle phosphorylase activity by carnosine and anserine

Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase $\text{a}$ in the presence and absence of AMP and phosphorylase $\text{b}$ in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase $\text{b}$ showing a greater degree of activation than phosphorylase $\text{a}$. Anserine (β-alanyl-L-N^π-methyl-histidine) activates phosphorylase $\text{a}$ to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase $\text{b}$ a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase $\text{a}$ in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase $\text{b}$ by anserine is also observed in the presence of these inhibitors.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

Action of 5-thio-D-glucose in the control of glycogen depolymerization

With muscle glycogen phosphorylase a and b, 5-thio-$\text{D}$-glucose is a non-competitive inhibitor toward phosphate where it has a K_i of 13 mM and 5.1 mM, respectively, and produces a mixed type of inhibition when glycogen is the substrate.5-Thio-$\text{D}$-glucose enhances diaphragm phosphorylase phosphatase activity to the same extent as $\text{D}$-glucose, yet the thioanalog does not affect phosphorylase b kinase. Thus, the action of 5-thio-$\text{D}$-glucose on glycogen degradation proceeds by inhibition of phosphorylase a and b and by inactivation of phosphorylase a through converting it to the b form.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases

This work describes the structure of a variety of lecithin-water phases observed below the “melting” temperature of the hydrocarbon chains, with special emphasis on the conformation of the chains. The lecithins studied in this work are the homologous series dioctanoyl to distearoyl, 2-decanoyl-1-stearoyl, and a preparation from hen eggs. The hydrocarbon chains are found to adopt a variety of conformations in addition to type α, the liquid-like organization observed above the melting temperature. Type β: the chains are stiff and parallel, oriented at right angles to the plane of the lamellae and packed with rotational disorder in a two-dimensional hexagonal lattice ($\text{a ~ 4.85}\text{A}\text{?}\text{)}$. Type β′: similar to β, but with the chains tilted with respect to the normal to the lamellae. Type δ: the chains are probably coiled into helices, whose axes are perpendicular to the plane of the polar groups and are packed with rotational disorder in a two-dimensional square lattice ($\text{a ~ 4.80}\text{A}\text{?}\text{)}$, α is the predominant conformation, common to most lipids in the presence of water and at sufficiently high temperature, and the one more relevant to membranes; β is observed at lower temperatures in lipids whose chains are heterogeneous and in the presence of very small amounts of water; β′ is found in synthetic lecithins with identical chains, in the presence of variable amounts of water; δ is observed in dry lecithins. A highly ordered crystalline phase, yet displaying rotational disorder of the chains, is observed in almost dry lecithins. Most of the phases are lamellar, and contain one lipid bilayer per repeat unit. Two phases display two-dimensional lattices: Pδ, formed by ribbon-like elements with the chains in the δ conformation; Pβ′, formed by lamellae of type β′ distorted by periodic ripples. The results emphasize the clear-cut difference between the liquid-like and the other types of partly ordered conformations, as well as the correlations which exist between the chemical composition and the structure of the lipids below the melting temperature of the chains.     

Substrate specificity of glycogen synthase phosphatase from bovine heart: action on phosphorylase a and histone

Glycogen synthase phosphatase has been purified from bovine heart. This preparation catalyzes conversion of synthase D into I and phosphorylase $\text{a}$ into $\text{b}$ and is able to dephosphorylate synthase D, phosphorylase $\text{a}$, active phosphorylase kinase, and phosphorylated histone and casein. The activity of phosphatase was assayed with synthase D, phosphorylase $\text{a}$, and histone as substrates after chromatography on Sephadex G-100, after sucrose gradient centrifugation, and after isoelectric focusing in a sucrose gradient. In all cases no separation of enzyme activity was observed with the above substrates. The phosphatase activity on all substrates was lost at the same rate by heat denaturation. These results indicate that this enzyme preparation contains a single phosphoprotein phosphatase which is responsible for the activity observed on the above substrates.     

Visualization of drug-nucleic acid interactions at atomic resolution: II. Structure of an ethidium/dinucleoside monophosphate crystalline complex,ethidium:5-iodocytidylyl (3′–5′) guanosine

Ethidium forms a second crystalline complex with the dinucleoside monophosphate 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, P2₁, with $\text{a = 14.06}\text{A}\text{?}\text{, b = 32.34}\text{A}\text{?}\text{, c = 16.53}\text{A}\text{?}\text{, β = 117.8 °}$. The structure has been solved to atomic resolution using rigid-body Patterson vector search and Fourier methods, and refined by full matrix least-squares to a residual of 0.16 on 3180 observed reflections. The structure consists of two ethidium molecules, two iodoCpG molecules, 27 water molecules and four methanol molecules, a total of 165 atoms (excluding hydrogens) in the asymmetric unit. Both iodoCpG molecules are hydrogen-bonded together by guanine · cytosine Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoCpG structure and between neighboring iodoCpG molecules in adjoining unit cells are separated by 6.7 Å. This distance reflects the presence of an ethidium molecule intercalated between base-paired iodoCpG molecules and another ethidium molecule stacked above (and below) the dinucleotide. Approximate 2-fold symmetry is used in the interaction; this reflects the pseudo-2-fold symmetry axis of the phenanthridinium ring system in ethidium coinciding with the approximate 2-fold axis relating base-paired iodoCpG molecules. The phenyl and ethyl groups of the intercalated ethidium molecule lie in the narrow groove of the miniature iodoCpG double-helix. The stacked ethidium, however, lies in the opposite direction, its phenyl and ethyl groups neighboring iodine atoms on cytosine residues. Base-pairs within the paired nucleotide units are related by a twist of about 8 °. The magnitude of this angular twist reflects conformational changes in the sugar-phosphate chains accompanying intercalation. These primarily reflect the differences in ribose sugar ring puckering that are observed (i.e. both iodocytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), and alterations in the glycosidic torsional angles that describe the base-sugar orientation.The information provided by this structure analysis (along with the accompanying one (ethidium:iodoUpA), described in the previous paper) has led to an understanding of the general nature of intercalative drug binding to DNA. This is described in the third paper of this series.     

Crystallisation of tRNALeuCUG from Escherichia coli after purification with hydroxyapatite.

tRNA_CUG^Leu from E. Coli was purified by column chromatography on benzoylated DEAE-cellulose, followed by hydroxyapatite prepared by an improved method. Crystals obtained by vapour diffusion gave X-ray diffraction out to 7 Å in the hk0 projection and 10 Å in h0?. The space group was P4₂2₁2 with $\text{a = b = 133}\text{A}\text{?}$, $\text{c = 66}\text{A}\text{?}$ and 8 molecules in the unit cell. Birefringence showed preferred orientation of RNA helical regions in the ab plane.     

Crystallographic study of the large tryptic fragments of elongation factor G from Escherichia coli

Crystals of N- and C-terminal fragments of elongation factor G (EF-G) from Escherichia coli have been grown from the preparations obtained by limited tryptic hydrolysis. Molecular masses of these fragments are equal to about 49,000 and 25,000, respectively. In the form of an additive complex they appear to be a major part of the native spatial structure of EF-G (Alakhov et al., 1979). Crystals of N-terminal fragment belong to space group P4₁2₁2 or P4₃2₁2 with unit cell dimensions $\text{a = b = 76.6}\text{A}\text{?}\text{and}\text{c = 191.6}\text{A}\text{?}$. Crystals of C-terminal fragment belong to space group P4₁22 or P4₃22 with unit cell dimensions $\text{a = b = 77.1}\text{A}\text{?}\text{and}\text{c = 75.0}\text{A}\text{?}$. In both cases the assumed number of protein molecules per asymmetric part of the unit cell is one.     

Crystallography of Bacillus subtilis levansucrase

Levansucrase, an exocellular enzyme, has been isolated from a high producer mutant, the BS5C4 constitutive strain, of Bacillus subtilis. Three crystalline forms have been obtained, all three belonging to the orthorhombic space group P2₁2₁2₁. The most suitable form for a three-dimensional structure investigation has cell dimensions, $\text{a = 68}\text{A}\text{?}$, $\text{b = 125}\text{A}\text{?}$, $\text{c = 54}\text{A}\text{?}$. There is one molecule in the asymmetric unit.     

Crystal structure of an inhibitor and a model for inhibition of replicative DNA synthesis

6-(p-Hydroxyphenylhydrazino)-uracil is an antimicrobial agent that selectively blocks replicative DNA synthesis in Bacillus subtilis by inhibiting DNA polymerase III. The drug crystallizes as a monoclinic monohydrate, space group $\text{C2}\text{c}$, with $\text{a = 23.920(6)}\text{A}\text{̊}\text{, b = 5.587(3)}\text{A}\text{̊}\text{, c = 17.466(5)}\text{A}\text{̊}\text{, β = 101.45(8) °}$, and eight hydrated molecules per cell. Three-dimensional X-ray diffraction data were collected. The structure was solved by Patterson methods and refined to an R value of 6.8% for the 1651 data. The geometry of the uracil ring is unusual. The bond distances suggest that a resonance form involving a positively charged hydrazino nitrogen and a negatively charged carbonyl oxygen, O(4), makes a large contribution to the valence bond structure of this compound. The exocyclic C(6)N bond is short (1.335 Å), the C(6)C(5) bond distance is 1.371 Å, which is longer than in uracil, and the C(5)C(4) distance (1.396 Å) is short. The uracil ring, the linked hydrazino nitrogen, and the hydrogen on this nitrogen are in the same plane. Each uracil group is hydrogen bonded to a nearly coplanar uracil across a center of symmetry. The water molecule is also near the plane of these paired bases and forms a hydrogen bond with the uracil-linked hydrazino NH group. This paired base arrangement and the restricted rotation about the exocyclic C(6)N link that constrains the hydrazino NH group to lie near the uracil plane suggest a model for the interaction of the drug with template-primer DNA. The drug acts when cytosine is the base to be copied in the template strand, and the drug is competitive with dGTP. Both cytosine and guanine can be accommodated with little distortion of the crystal structure geometry in a manner compatible with the known geometry of DNA. The structural and biochemical aspects of the model for drug action are discussed.     

Nitration of functional tyrosyl residues in rabbit muscle phosphorylase b

Upon reaction of rabbit muscle phosphorylase $\text{b}$ with tetranitromethane in a stoichiometric ratio with respect to the tyrosyl content, 2 out of 34 phenolic groups per mole of monomer (M.W. 95,000) were nitrated with an almost complete loss of activity. Only one residue per monomer was nitrated in the presence of AMP, the major part of the activity being preserved. The sedimentation pattern of modified phosphorylase $\text{b}$ showed that, following nitration in the absence of AMP, the enzyme was fully dissociated into monomers, whereas, when the enzyme was nitrated in its presence, the dimeric structure was retained.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

X-ray study on egg-yolk lecithin: Unit cell data and electron density profile

The unit cell dimensions and space group of egg-yolk lecithin (l-α-phosphatidylcholine) in the crystalline state were obtained from a set of X-ray fibre diagrams recorded from specimens oriented in two different axes; $\text{a = 8.84}\text{A}\text{?}\text{± 0.05}\text{A}\text{?}\text{, b = 10.07}\text{A}\text{?}\text{± 0.05}\text{A}\text{?}\text{, c = 54.69}\text{A}\text{?}\text{± 0.05}\text{A}\text{?}$, with space group P2₁2₁2, and four molecules per unit cell. From the 00l reflexions up to the 25th order, an electron density profile is obtained which is consistent with a probable structure of the molecule.