Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gel: Application to cationic proteins in serum

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gels have been developed for the analysis of the cationic proteins in complex mixtures of protein. A pH gradient covering the “entire” pH range but emphasizing only a narrow portion of this gradient is produced. During the first stage a nonlinear pH gradient is nearly established. The anodic electrode is then moved to a position on the gel such that the applied potential is exclusively across the alkaline portion of the gradient. The second stage results in a significant increase in the electric field strength and, consequently, the resolving power. The two-stage methods, exploiting the advantages of narrow pH gradients and high field strengths, have enabled excellent resolution of the highly heterogeneous cationic proteins in serum.     

Isolation and characteristics of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Phospholipase A₂ was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 20,000. The optimum pH and temperature of the enzyme were at around pH 9.0 and 50°C, respectively, and the activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The enzyme had no fatty acid specificity. Starfish phospholipase A₂ hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine.     

Polymorphism of vitamin B12 [57Col binding proteins in rabbit serum

When rabbit serum labelled with vitamin B₁₂[⁵⁷Co] was subjected to starch gel electrophoresis and au;oradiography, three phenotypes of proteins capable of binding vitamin B₁₂ were observed. Family data revealed that these phenotypes (called TC-A, TC-AB and TC-B) are controlied by two codominant alleles (TC^A and TC^B), at an autosomic locus. Proteins capable of binding vitamin B₁₂ both in vivo and in vitro are commonly referred to as Transcobalamins and can be found in the serum of numerous animal species (for a review, see Glass, 1974; Allen, 1975; Stenman, 1975). Furthermore, Daiger et al. (1975a) have described seven different patterns of vitamin B₁₂ binding proteins which occur in human plasma and which are presumably controlled by four alleles. The present paper describes experiments in which both starch gel electrophoresis and autoradiography are used to identify three phenotypes of rabbit serum proteins responsible for binding vitamin B₁₂ in vitro. It was found that these three phenotypes are controlled by two allelic codominant genes, at an autosomic locus. Individual serum samples (30 μl), obtained from 385 White New Zealand rabbits varying in age from one month to three years, were incubated with 0.1 ng of vitamin B₁₂[⁵⁷Co] (specific activity: 180 μCi/μg; Lot 247; Radiochemical Centre, Amersham, England) at 37°C for 30 minutes. Starch gel electrophoresis and autoradiography were performed as described by Geldermann (1970) and Daiger et al. (1975b), respectively. Electrofocusing (pH range 3.5–9.5) was conducted in the 2117 Multiphor apparatus (LKB, Bromma, Sweden) according to the manufacturer's instructions. The resulting pH gradient was measured with a surface pH electrode (Ingold, Zürich, Switzerland).     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Circular Dichroism and Fluorescence Studies of Polyomavirus Major Capsid Protein VP1

The conformational changes of polymavirus (Py) major capsid protein VP₁ in solution by the solution pH, addition of calcium, and ionic strength were examined by circular dichroism (CD) and fluorescence spectroscopy. Comparison of the predicted secondary structures of PyVP₁ and simian virus (SV) 40 by the methods of Chou-Fasman, Gamier et al., and Yang method are presented. Hydropathicity, surface probability, and chain flexibility of PyVP₁ were computer-analyzed by the methods of Kyte and Doolittle, Emini et al., and Karplus and Schulz, respectively. The CD measurements indicate that the secondary structure of PyVP₁ is little dependent on its concentration, Ca²⁺ concentration, and ionic strength, but is strongly pH dependent. Fluorescence studies showed that emission spectra of PyVP₁ are also pH-dependent. At extreme acidic and alkaline pH, the fluorescence intensity of PyVP₁ is decreased and the emission maximum is red-shifted. The fluorescence of PyVP₁ is quenched by the presence of CsCl, KI, and acrylamide. The analyses of the modified Stern–Volmer plots indicate that five of seven tryptophan residues in PyVP₁ are located on the surface of the protein, among which two are accessible to Cs⁺ and the other three are accessible to I^–. The two others are buried more deeply in the interior of the protein molecule.On leave from National Taiwan University;     

Effect of pH on vitamin B12 binding by transcobalamins

An investigation has been made of the effect of varying pH at constant ionic strength on vitamin B₁₂ binding by human serum and by two transcbalamin fractions separated from serum by gel filtration. It was found that the methodology used had a considerable influence on the results obtained. Genuine effects of pH were largely confined to reduced vitamin B₁₂ binding at very acid and very alkaline pH. However, due to an adsorption artefact involving transcobalamin II, certain methods appeared to demonstrate a marked decrease in vitamin B₁₂ binding between pH 4.5 and pH 10.3, especially in the range of pH 5.3–7.5.     

Acclimation of photosynthesis to elevated CO2 in onion (Allium cepa) grown at a range of temperatures

Onion (Allium cepa) was grown in the field within temperature gradient tunnels (providing about ‐2.5°C to +2.5°C from outside temperatures) maintained at either 374 or 532 μmol mol^?1 CO₂. Plant leaf area was determined non‐destructively at 7 day intervals until the time of bulbing in 12 combinations of temperature and CO₂ concentration. Gas exchange was measured in each plot at the time of bulbing, and the carbohydrate content of the leaf (source) and bulb (sink) was determined. Maximum rate of leaf area expansion increased with mean temperature. Leaf area duration and maximum rate of leaf area expansion were not significantly affected by CO₂. The light‐saturated rates of leaf photosynthesis (A_sat) were greater in plants grown at normal than at elevated CO₂ concentrations at the same measurement CO₂ concentration. Acclimation of photosynthesis decreased with an increase in growth temperature, and with an increase in leaf nitrogen content at elevated CO₂. The ratio of intercellular to atmospheric CO₂ (C₁/C₃ ratio) was 7.4% less for plants grown at elevated compared with normal CO₂. A_sat in plants grown at elevated CO₂ was less than in plants grown at normal CO₂ when compared at the same C₁. Hence, acclimation of photosynthesis was due both to stomatal acclimation and to limitations to biochemical CO₂ fixation. Carbohydrate content of the onion bulbs was greater at elevated than at normal CO₂. In contrast, carbohydrate content was less at elevated compared with normal CO₂ in the leaf sections in which CO₂ exchange was measured at the same developmental stage. Therefore, acclimation of photosynthesis in fully expanded onion leaves was detected despite the absence of localised carbohydrate accumulation in these field‐grown crops.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

The effect of orally administered prostaglandins A1, A2 and 15 Epi-A2 on human gastric acid secretion

Prostaglandins A₁, A₂ and 15 Epi-A₂ were administered orally to human male volunteers. Prostaglandin A₁ and 15 Epi-A₂ did not consistently affect gastric acid secretion either in terms of the pH values within individual tests or in respect of comparative control test data. Prostaglandin A₂ administration resulted in a transient inhibition of secretion in all 6 subjects tested, with the pH rising above 6 in every case.It is concluded that none of these compounds is likely to have therapeutic application to the peptic ulceration problem.     

Isoelectric focusing as the crow flies

The evolution of isoelectric focusing is traced back over the years, from a somewhat shaky origin to present-day immobilized pH gradients. Four generations of methodology are classified and discussed: (A) Kolin's approach, consisting of a two-step technique, generation of a pH gradient by diffusion followed by a rapid electrokinetic protein separation; (B) Svensson-Rilbe's approach, consisting of creating a pH gradient in an electric field by utilizing as buffers a multitude of carrier ampholytes, i.e. of amphoteric species possessing good buffering capacity and conductivity at their pI; (C) immobilized pH gradients, by which non-amphoteric buffers and titrants (acrylamido weak acids and bases), titrated around their pK values, are grafted (insolubilized) onto a polyacrylamide gel matrix and (D) mixed-bed carrier ampholyte-Immobiline gel, by which a soluble, carrier ampholyte generated pH gradient coexists in the same matrix with an insoluble, Immobiline generated, pH gradient.     

Lipid phase separation mediates binding of porcine pancreatic phospholipase A2 to its substrate

Multilamellar liposomes made of equimolar mixtures of dimyristoyl and distearoylphosphatidylcholine were hydrolysed by porcine pancreatic phospholipase A₂. Ph-stat titration, equilibrium gel filtration and differential scanning calorimetry were used to study respectively the enzymic hydrolysis, the enzyme binding and the lipid phase repartition. We demonstrated that the optimal enzyme activity observed in the region where liquid crystalline and gel lipid phases coexist, is due to a drastic increase of the enzyme binding to its substrate. It is suggested that the border region separating the lipid phases could be a privileged site for enzyme insertion. Increase of lateral compressibility due to coexistence of solid and fluid lipid phases will promote the penetration of the hydrophobic interface recognition site (IRS) of phospholipase A₂ into the lipid matrix whereas the active site, distinct from the IRS will attack its substrate independently of the lipid physical state.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

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

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

Canopy gradients in leaf intercellular CO2 mole fractions revisited: interactions between leaf irradiance and water stress need consideration

Intercellular CO₂ mole fractions (C_i) are lower in the upper canopy relative to the lower canopy leaves. This canopy gradient in C_i has been associated with enhanced rates of carbon assimilation at high light, and concomitant greater draw‐downs in C_i. However, increases in irradiance in the canopy are generally also associated with decreases in leaf water availability. Thus, stress effects on photosynthesis rates (A) and stomatal conductance (G), may provide a further explanation for the observed C_i gradients. To test the hypotheses of the sources of canopy variation in C_i, and quantitatively assess the influence of within‐canopy differences in stomatal regulation on A, the seasonal and diurnal variation in G was studied in relation to seasonal average daily integrated quantum flux density (Q_int) in tall shade‐intolerant Populus tremula L. trees. Daily time‐courses of A were simulated using the photosynthesis model of Farquhar et al. (Planta 149, 78–90, 1980). Stable carbon isotope composition of a leaf carbon fraction with rapid turnover rate was used to estimate canopy gradient in C_i during the simulations. Daily maximum G (G_max) consistently increased with increasing Q_int. However, canopy differences in G_max decreased as soil water availability became limiting during the season. In water‐stressed leaves, there were strong mid‐day decreases in G that were poorly associated with vapour pressure deficits between the leaf and atmosphere, and the magnitude of the mid‐day decreases in G occasionally interacted with long‐term leaf light environment. Simulations indicated that the percentage of carbon lost due to mid‐day stomatal closure was of the order of 5–10%, and seasonal water stress increased this percentage up to 20%. The percentage of carbon lost due to stomatal closure increased with increasing Q_int. Canopy differences in light environment resulted in a gradient of daily average C_i of approximately 20 µmol mol^?1. The canopy variation in seasonal and diurnal reductions in G led to a C_i gradient of approximately 100 µmol mol^?1, and the actual canopy C_i gradient was of the same magnitude according to leaf carbon isotope composition. This study demonstrates that stress effects influence C_i more strongly than within‐canopy light gradients, and also that leaves acclimated to different irradiance and water stress conditions may regulate water use largely independent of foliar photosynthetic potentials.     

Gas exchange and photosynthetic acclimation over subambient to elevated CO2 in a C3–C4 grassland

Atmospheric CO₂ (C_a) has risen dramatically since preglacial times and is projected to double in the next century. As part of a 4‐year study, we examined leaf gas exchange and photosynthetic acclimation in C₃ and C₄ plants using unique chambers that maintained a continuous C_a gradient from 200 to 550 µmol mol^?1 in a natural grassland. Our goals were to characterize linear, nonlinear and threshold responses to increasing C_a from past to future C_a levels. Photosynthesis (A), stomatal conductance (g_s), leaf water‐use efficiency (A/g_s) and leaf N content were measured in three common species: Bothriochloa ischaemum, a C₄ perennial grass, Bromus japonicus, a C₃ annual grass, and Solanum dimidiatum, a C₃ perennial forb. Assimilation responses to internal CO₂ concentrations (A/C_i curves) and photosynthetically active radiation (A/PAR curves) were also assessed, and acclimation parameters estimated from these data. Photosynthesis increased linearly with C_a in all species (P < 0.05). S. dimidiatum and B. ischaemum had greater carboxylation rates for Rubisco and PEP carboxylase, respectively, at subambient than superambient C_a (P < 0.05). To our knowledge, this is the first published evidence of A up‐regulation at subambient C_a in the field. No species showed down‐regulation at superambient C_a. Stomatal conductance generally showed curvilinear decreases with C_a in the perennial species (P < 0.05), with steeper declines over subambient C_a than superambient, suggesting that plant water relations have already changed significantly with past C_a increases. Resource‐use efficiency (A/g_s and A/leaf N) in all species increased linearly with C_a. As both C₃ and C₄ plants had significant responses in A, g_s, A/g_s and A/leaf N to C_a enrichment, future C_a increases in this grassland may not favour C₃ species as much as originally thought. Non‐linear responses and acclimation to low C_a should be incorporated into mechanistic models to better predict the effects of past and present rising C_a on grassland ecosystems.     

Charged acrylamide copolymer gels as media for weak alignment

The use of mechanically strained acrylamide/acrylate copolymers is reported as a new alignment medium for biomacromolecules. Compared to uncharged, strained polyacrylamide gels, the negative charges of the acrylamide/acrylate copolymer strongly alter the alignment tensor and lead to pronounced electroosmotic swelling. The swelling itself can be used to achieve anisotropic, mechanical strain. The method is demonstrated for the alignment of TipAS, a 17 kDa antibiotic resistance protein, as well as for human ubiquitin, where alignment tensors with an A_ZZ,NH of up to 60 Hz are achieved at a gel concentration of 2% (w/v). The alignment can be modulated by the variation of pH, ionic strength, and gel concentration. The high mechanical stability of the swollen gels makes it possible to obtain alignment at polymer concentrations of less than 1% (w/v).     

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.