High-performance liquid chromatographic assay of l-α-glycerophosphorylcholine using a two-step enzymic conversion

A high-performance liquid chromatographic method using an enzymic reactor for determination of l-α-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of l-α-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml l-α-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 μl of injected sample. The precision of the method in the analysis of l-α-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

A new method for the demonstration of phase transitions in lipid films

Monolayers of l-α-lecithin (β,γ-dimyristoyl), l-α-lecithin (β,γ-dipalmitoyl) and egg phosphatidylethanolamine were formed on a water surface at a sufficiently low temperature. The rate of evaporation of water was measured as a function ofincreasing temperature.In agreement with previous reports, the monomolecular films had a marked influence on the rate of evaporation of water, and the passage of water molecules through the monolayer was retarded.Using films in which phase transitions can be observed at a specific transition temperature [l-α-lecithin (β,γ-dimyristoyl) at 23°C and l-α-lecithin (β,γ-dipalmitoyl) at 41 °C], an unexpected decrease of water permeability was measured at this particular temperature. The retardation suggests that water transport through the film is hindered. It is thought that these results are best explained by an enhanced interaction between the water molecules and the chains of the lipid molecules above the phase transition temperature.     

A P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.     

1-β- -Arabinofuranosylcytosine stimulates thymidine incorporation into the DNA of contact-inhibited cells

In rapidly proliferating cells l-β- -arabinofuranosylcytosine (ara-C) is a potent inhibitor of DNA synthesis whose effect can be irreversible and consequently cytocidal. Whereas thymidine incorporation is greatly reduced in rapidly proliferating cells in the presence of ara-C, contact-inhibited cells, similarly treated, show increased thymidine incorporation by as much as 7-fold. This ara-C-induced stimulation appears to result from an influence on thymidine utilization rather than increased DNA synthesis.     

Acid glycosidases from hen oviduct and egg albumen

Activities of seven acid glycosidases: β-N-acetylhexosaminidase (β-HEX), α- and β-galactosidase (α- and β-GAL), α- and β-mannosidase (α- and β-MAN), α-glucosidase and α-fucosidase in magnum region of hen (Gallus gallus domesticus) oviduct, and four acid glycosidases: β-HEX, β-GAL, α- and β-MAN in egg albumen, were investigated. β-HEX from magnum and egg albumen hydrolysed 4-methylumbelliferyl-β-N-acetylhexosamine-6-sulphate (4-MeUmbGlcNAc-6-SO₄) like mammalian β-HEX form A. Multiple forms of magnum and egg albumen β-HEX, β-GAL, α- and β-MAN were separated by strong anion exchange chromatography and chromatofocusing method. Chromatofocusing of the magnum resulted in the appearance of multiple forms for β-HEX with pI of 6.18, 5.43, 5.55, 5.34, 5.27 and 5.16, for β-GAL with pI of 4.98, 4.84, 4.77, 4.64 and 4.68–4.63, for α-MAN with pI of ≥ 7.4, 6.75, 6.62 and 6.26, and for β-MAN two forms with pI of 6.37 and 5.77. Chromatofocusing of egg albumen yields multiple forms for β-HEX with pI of 6.24, 6.08, 5.55 and 5.35, for β-GAL two forms with pI of 5.10 and 4.86–4.80 for α-MAN multiple forms with pI of ≥ 7.4, 6.80, 6.60 and 6.30, and for β-MAN forms with pI of 6.30 and 5.77. In conclusion, this study was the first to show β-HEX activity against 4-MeUmbGlcNAc-6-SO₄ in the magnum and albumen of bird eggs, corresponding to β-HEX A activity in mammals. Main multiple forms of β-HEX, β-GAL, α- and β-MAN occurring in the magnum were revealed in the egg albumen. Comparison with a cock of the same breed showed that hen egg magnum and albumen has the same multiple forms of the enzymes that are found in the epididymides and seminal plasma of the cock.     

Flavin-linked mitochondrial α-glycerophosphate dehydrogenase of Candida utilis

The 150-fold purification of the l-α-glycerophosphate dehydrogenase of Candida utilis electron-transport particles by very mild procedures is described. The active enzyme contains FAD, iron and copper. The function of the metals, if any, is not clear. Its molecular weight is about 5·10⁵. The subunit composition is complex and remains unresolved because the enzyme is contaminated with protease(s). The activity of this enzyme is very low in Saccharomyces cerevisiae unless the cells are grown in glycerol. The NAD-dependent cytoplasmic α-glycerophosphate dehydrogenase is present in C. utilis but could not be demonstrated in glucose-grown S. cerevisiae.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

A mechanism for suppression of the CDP-choline pathway during apoptosis

Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-deficient MCF7 cells to show that caspase 3 is required for CCTα proteolysis and release from the nucleus during apoptosis. CCTα-Δ28 (a caspase-cleaved mimic) expressed in CCTα-deficient Chinese hamster ovary cells was cytosolic and had increased in vitro activity. However, [³H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCTα. In camptothecin-treated MCF7 and MCF7-C3 cells, choline kinase activity was unaffected; however, choline transport into cells was reduced by 30 and 60%, respectively. We conclude that caspase 3-mediated removal of the CCTα NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. Rather, the CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.     

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1β and a protein kinase C activator

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1β (rIL-1β) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1β alone at concentrations of 1–100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1β (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1β or PKC agonists alone or in combination. Recombinant tumor necrosis factor α (rTNFα) did not substitute for IL-1β in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1β or TNFα alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.     

Identification of α- and β-species of calcitonin gene-related peptide in the rat amygdala after separation with capillary zone electrophoresis

Calcitonin gene-related peptide (CGRP) may be present in two forms in nervous tissue. Reversed-phase high-performance liquid chromatography has previously been found to be insufficient to clearly separate α-CGRP and β-CGRP. A method for the separation of CGRPs by capillary zone electrophoresis has been developed. Separation of human or rat α-CGRP and β-CGRP was achieved at pH values between 3.5 and 4.5 and a potential of 20 kV in a fused-silica capillary. Electrophoresis of an extract of rat amygdala in a micropreparative way, with subsequent radioimmunoassay, revealed for the first time the presence of α-CGRP and β-CGRP in this brain area. The method may thus be used for separation of CGRPs, to reveal the distribution of α-CGRP and β-CGRP, and for purity control.     

Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using β-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major α-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of α-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the β-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.

Evidence showing that β-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.

     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Enzymic radioassay for acetylcholine and choline in brain

This assay for acetylcholine (ACh) or choline in extracts of rat brain involves the isolation of the choline ester by high-voltage paper electrophoresis, alkaline hydrolysis of ACh to choline, and the quantitative enzymic conversion of choline to a radioactive derivative, P³²-phosphorylcholine. The method is specific, is applicable to large numbers of tissue samples, and has a blank value of about 3 nanograms of choline.     

Choline and Choline Metabolite Patterns and Associations in Blood and Milk during Lactation in Dairy Cows

Milk and dairy products are an important source of choline, a nutrient essential for human health. Infant formula derived from bovine milk contains a number of metabolic forms of choline, all contribute to the growth and development of the newborn. At present, little is known about the factors that influence the concentrations of choline metabolites in milk. The objectives of this study were to characterize and then evaluate associations for choline and its metabolites in blood and milk through the first 37 weeks of lactation in the dairy cow. Milk and blood samples from twelve Holstein cows were collected in early, mid and late lactation and analyzed for acetylcholine, free choline, betaine, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine and sphingomyelin using hydrophilic interaction liquid chromatography-tandem mass spectrometry, and quantified using stable isotope-labeled internal standards. Total choline concentration in plasma, which was almost entirely phosphatidylcholine, increased 10-times from early to late lactation (1305 to 13,535 µmol/L). In milk, phosphocholine was the main metabolite in early lactation (492 µmol/L), which is a similar concentration to that found in human milk, however, phosphocholine concentration decreased exponentially through lactation to 43 µmol/L in late lactation. In contrast, phosphatidylcholine was the main metabolite in mid and late lactation (188 µmol/L and 659 µmol/L, respectively), with the increase through lactation positively correlated with phosphatidylcholine in plasma (R ² = 0.78). Unlike previously reported with human milk we found no correlation between plasma free choline concentration and milk choline metabolites. The changes in pattern of phosphocholine and phosphatidylcholine in milk through lactation observed in the bovine suggests that it is possible to manufacture infant formula that more closely matches these metabolites profile in human milk.     

Some structural features of the -glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a -glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 -glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the -glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked -glucosyl residues in a sequence. Both α- and β- -glucosidic linkages are present in the polysaccharide, the former preponderating. The -glucan gave with iodine a faint blue color that had λ_max 420 nm.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.