Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A₂ activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from $\text{E.}$$\text{coli}$ membranes. Phospholipase A₂ activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A₂ activity which was assayable in islet homogenates. Glucose stimulated phospholipase A₂ in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A₂ activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A₂ activity measured in the presence of high calcium concentrations. Phospholipase A₂ activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A₂ activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.     

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce more TxA₂ than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI₂) than arterial tissue from vitamin E-supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2, 6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10 s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light → A₀-light → A₁-dark → A₂-light → A₃ in which P is a precursor convertible to A₀, the first intermediate with a longer lifetime of $\text{t}\text{1}\text{2}\text{≈ 100}\text{s}$ and A₃ is the final activated compound or state converted by short-interval flashes from A₀ through A₁ and A₂, two other intermediates with shorter lifetimes of $\text{t}\text{1}\text{2}\text{≈ 0.4}\text{s}$ and 5 s, respectively.     

PGE2 and PGF2α biosynthesis in stimulated and nonstimulated peritoneal preparations containing macrophages

The biosynthesis of PGE₂ and PGF_2α was measured in intact peritoneal exudate preparations obtained fom $\text{C. parvum}$-treated and control C₃H mice. Although both the control and stimulated preparations biosynthesized PGF_2α and PGE₂ from [1–14C] arachidonic acid, the stimulated preparations generated more of both prostaglandins than did nonstimulated preparations, probably as a result of increased synthesis within macrophages. Increased transformation of PGE₂ into PGF_2α 9-ketoreductase was noted in stimulated preparations when compared to that in control cells. The data suggest that stimulated macrophages are capable of generating increased quantities of PGF_2α and therefore might function as one source of this substance in resolving inflammatory reactions.     

Solubilization of the opiate receptor

The opiate receptor is solubilized from rat neural membranes by treating the membranes with Triton X-100, followed by centrifugation. Removal of the Triton X-100 was accomplished with Bio-beads SM-2, and the resulting supernatant was capable of stereospecifically binding opiates at 10^?13 moles/mg protein under saturating conditions. Stereospecific binding was measured by equilibrium dialysis and gel filtration using a Sephadex G-25 column, equilibrated with [³H] -ligand and either dextrorphan or levorphanol. The solubilized receptor has affinities for the opiates similar to those observed in membrane preparations and $\text{in vivo}$ experiments. The addition of phosphatidylserine to the supernatant enhances stereospecific binding of etorphine slightly. Phospholipase A₂, trypsin and chymotrypsin completely inhibit binding. The addition of albumin prevents, but does not reverse the inhibition caused by low concentrations of phospholipase A₂. Phosphatidylserine decarboxylase inhibits stereospecific binding by 95%, despite the fact only 10% of the phosphatidylserine present in the supernatant is converted to phosphatidylethanolamine. The solubilized opiate receptor, like the receptor in neural membranes, appears to consist of both protein and lipid moieties.     

Pancreatic phospholipase A2 is not regulated by calmodulin

It was previously suggested [Wong, P.Y.-K and Cheung, W.Y. (1979) Biochem. Biophys. Res. Comm. $\text{90}$, 473–480] that the Ca²⁺ activation of phospholipase A₂ is mediated by the calcium binding protein calmodulin. In the present study phospholipase A₂ from pig pancreas was shown to be absolutely Ca²⁺ dependent but the enzyme was not stimulated by exogenous calmodulin and no endogenous calmodulin was found in the preparation. The enzyme was inhibited in the absence of calmodulin by several drugs (trifluoperazine, mepacrine, promethazine and propranolol) which are known to bind to calmodulin. A kinetic analysis indicated that trifluoperazine competitively inhibited phospholipase A₂, probably by interacting with phospholipid substrate.     

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.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

Manganese superoxide dismutases from Escherichia coli and from yeast mitochondria: Preliminary X-ray crystallographic studies

The Mn superoxide dismutase from Escherichia coli has been obtained in three crystal forms: (I) from 68% saturated (NH₄)₂SO₄, space group P222 or P222₁, $\text{a = 47}\text{A}\text{?}\text{, b = 103}\text{A}\text{?}\text{, c = 47.5}\text{A}\text{?}$, with one subunit per asymmetric unit; (II) from 50% polyethylene glycol 6000, space group C222₁ (with approx. P4₁2₁2 symmetry), $\text{a = 101}\text{A}\text{?}\text{, b = 108}\text{A}\text{?}\text{, c = 180}\text{A}\text{?}$, with four subunits (2 molecules) per asymmetric unit; (III) from 52% polyethylene glycol with a different method of preparing the enzyme solution, space group P2₁2₁2, $\text{a = 47}\text{A}\text{?}\text{, b = 51}\text{A}\text{?}\text{, c = 188}\text{A}\text{?}$, with two subunits per asymmetric unit.The yeast mitochondrial Mn superoxide dismutase has yielded the same crystal form both from 30% 2-methyl-2,4-pentane diol and from 23% polyethylene glycol 6000: space group P2₁2₁2₁, $\text{a = 63}\text{A}\text{?}\text{, b = 115}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, with four subunits (one molecule) per asymmetric unit.A full X-ray crystallographic study of at least one of these enzymes is planned.     

Preliminary crystallographic data for lysozyme produced by Streptomyces erythraeus.

Crystals of lysozyme from Streptomyces erythraeus are orthorhombic, space group P2₁2₁2₁ with unit cell dimensions: $\text{a = 50.01}\text{A}\text{?}\text{, b = 61.98}\text{A}\text{?}$ and $\text{c = 67.85}\text{A}\text{?}$; and one molecule, of molecular weight about 18,500, per asymmetric unit.     

Letter: Crystallographic study of an esteroproteolytic enzyme

The esteroproteolytic enzyme, a serine protease from porcine pancreas, has been crystallized and characterized by X-ray diffraction. Two closely related crystal forms were observed. The unit cell dimensions of the first form are $\text{a = 59.2}\text{A}\text{?}\text{, b = 96.4}\text{A}\text{?}\text{and}\text{c = 47.4}\text{A}\text{?}$, space group P2₁2₁2 with one molecule (mol. wt 30,000) per asymmetric unit. The unit cell dimensions of the second form are $\text{a = 59.2}\text{A}\text{?}\text{, b = 96.4}\text{A}\text{?}\text{and}\text{c = 94.8}\text{A}\text{?}$, space group P2₁2₁2₁. Mercury derivatives of enzyme inhibitors were used to obtain multiple isomorphous heavy-atom substituents suitable for phase determination.     

Crystallization and preliminary crystallographic data for cytochrome c551.5 (c7) from Desulfuromonas acetoxidans.

Cytochrome c₇, a low potential, three heme-containing protein (molecular weight 9800) was isolated from Desulfuromonas acetoxidans and crystallized from polyethylene glycol solutions. The crystals belong to the orthorhombic space group P2₁2₁2₁, with unit-cell dimensions $\text{a = 44·10}\text{A}\text{?}\text{, b = 37·55}\text{A}\text{?}$ and $\text{c = 45·08}\text{A}\text{?}$ and have one molecule of protein per asymmetric unit.     

Structural study of crystalline auromomycin: Preliminary X-ray diffraction study of auromomycin and macromomycin

Auromomycin and macromomycin from the organism Streptomyces macromomyceticus have been crystallized. The X-ray diffraction pattern of crystals of each molecule is consistent with space group P2₁2₁2 with cell parameters $\text{a = 46.45}\text{A}\text{?}\text{, b = 54.34}\text{A}\text{?}\text{and}\text{c = 42.03}\text{A}\text{?}$ for auromomycin, and $\text{a = 46.45}\text{A}\text{?}\text{, b = 54.52}\text{A}\text{?}\text{and}\text{c = 41.54}\text{A}\text{?}$ for macromomycin. Diffraction analysis of auromomycin is in progress.     

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C₁₂-subunit of the previously determined partial structure $\text{2a}$ (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C₁₂-substructure, whereas a parallel two-step reduction occurs on NaBH₄ treatment. Both reactions occur with rearrangement of the C₁₂-substructure and the implication for the mechanism of action of NCS-Chrom $\text{A}$ in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom $\text{A}$ as well as minor components $\text{B}$ and $\text{C}$ by two protons.     

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.     

Crystallization of the Arc repressor

Arc, a represser from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P2₁2₁2₁ with $\text{a = 90.26}\text{A}\text{?}\text{, b = 52.88}\text{A}\text{?}\text{and}\text{c = 47.58}\text{A}\text{?}$. The crystals diffract to 2.2 Å resolution.