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.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C₆-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A₂ (PA₂) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C₆-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca⁺⁺ was required for PA₂ catalysis. Identification of the products of hydrolysis showed cleavage at the $\text{sn}$-1 and $\text{sn}$-2 positions for LpL and PA₂, respectively. For PA₂, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.     

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.     

Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes

A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3′,5′-cyclic nucleotide phosphodiesterase, and $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A₂ attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A₂ was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3′,5′-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500–4000Å), which do not form vesicles.     

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.     

Crystallographic study of beef liver catalase

An orthorhombic form of beef liver catalase crystals has been examined by X-ray diffraction and electron microscopy. The space group is P2₁2₁2₁ with $\text{a = 89}\text{A}\text{?}\text{, b = 140}\text{A}\text{?}\text{, and}\text{c = 231}\text{A}\text{?}$. Electron micrographs show the presence of broad channels arrayed in a hexagonal manner. The solvent content of these orthorhombic crystals is considerably less than that for previously reported trigonal crystals of beef liver catalase although the packing arrangement appears to be virtually the same, suggesting a possible symmetry transition upon dehydration.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

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.     

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.     

Role of the plasma membrane in the mechanism of cholesterol efflux from cells

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [³H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu₅AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for plasma membrane vesicles of Fu₅AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu₅AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu₅AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for cholesterol efflux from these cell lines.     

A turn in the road: How studies on the pharmacology of glucosylceramide synthase inhibitors led to the identification of a lysosomal phospholipase A2 with ceramide transacylase activity

A series of inhibitors of glucosylceramide synthesis, the PDMP based family of compounds, has been developed as a tool for the study of sphingolipid biochemistry and biology. During the course of developing more active glucosylceramide synthase inhibitors, we identified a second site of inhibitory activity for PDMP and its structural homologues that accounted for the ability of the inhibitors to raise cell and tissue ceramide levels. This inhibitory activity was directed against a previously unknown pathway for ceramide metabolism, viz. the formation of 1-O-acylceramide. In this pathway the addition of a fatty acyl group to the primary hydroxyl of ceramide occurs through a transacylation with either phosphatidylethanolamine or phosphatidylcholine as a substrate. However, both in the absence and presence of ceramide, water serves as an acceptor for the fatty acid. Thus the enzyme may be considered to be a phospholipase A₂. The enzyme is unique in that it has an acidic pH optimum and is localized to lysosomes by cell fractionation. More recently, the 1-O-acylceramide synthase has been purified, sequenced, and cloned. This phospholipase A₂ was discovered to be structurally homologous to lecithin cholesterol acyltransferase (LCAT). However, this phospholipase A₂ does not recognize cholesterol and lacks the defined lipoprotein-binding domain present in LCAT. We now refer to this enzyme as lysosomal phospholipase A₂ (LPLA₂). Although acidic phospholipase A₂ activities have been previously identified, LPLA₂ appears to be the first lysosomal PLA₂ to have been sequenced. This new phospholipase A₂ lacks an obvious and proven biological function. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin

The human erythrocyte membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase responded to the presence of an acidic phospholipase A₂ and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the $\text{Ca}{}^{\mathrm{2+}}\text{M}{}^{\mathrm{2+}}$ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane

After incubation of human erythrocytes at 37 °C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A₂ from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes.Pancreatic phospholipase A₂, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, $\text{o-}\text{iodosobenzoate}$ and hydroquinone) even render susceptible to pancreatic phospholipase A₂ phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Structure of Bence--Jones protein, Pav: an initial report

A Bence-Jones protein, Pav, was isolated from the urine of a myeloma patient. Crystals were grown by five different methods yielding different morphologies with slightly changed cell parameters, but the space group was the same (P2₁2₁2₁) in every case and X-ray patterns appeared to be identical. The cell parameters are: $\text{a = 93.6 (4)}\text{to}\text{95.1(4)}\text{A}\text{?}\text{, b = 92.7(3)}\text{A}\text{?}\text{and}\text{c = 72.8(2)}\text{A}\text{?}$. The crystal density and solvent content are approximately 1.128 g/cm³ and 0.64, respectively. Chemical evidence suggest that the two subunits of Pav are identical in chemical sequence. The crystal structure may prove useful in defining allosteric effects among antibody domains.     

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.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.