Biospecific chromatography of lipolytic enzymes and methods for determining their activity

The review concerns application of affinity chromatography for isolation of phospholipases and lipases, as well as the methods for determining their activities. Main emphasis is laid on the preparation of biospecific supports with lipid ligands as well as on development of new methods for assaying lipolytic activity.     

Synthesis of leukotriene A4 methyl ester via acetylene intermediates

Rac-leukotriene A4 methyl ester has been synthesized from propargylic alcohol and 1-heptyne. The synthetic strategy involves the assembly of carbon chain by acetylenide anion condensations and the introduction of (Z)-double bonds by the triple bond hydrogenation.     

Synthesis of phosphonate analogs of sphingomyelins and preparation of an affinity sorbent for sphingomyelinase purification

Phosphonate analogues of 2-N-stearoyl- (I) and 2-N-(undec-10-enoyl)-sphingomyelins (II) have been synthesised. Compound (II) was used as a starting product for preparation of a sorbent for sphingomyelinase affinity chromatography. The double bond of the unsaturated undec-10-enoyl moiety of the phosphonate analogue (II) was oxidized, and the modified (II) was coupled to amino-Toyopearl HW-65 to give a sorbent containing 4 mumoles of ligand per milliliter of the swollen resin.     

Two forms of glutamine synthetase from pumpkin leaves

Two molecular forms of glutamine synthetase localized in the cytoplasm and in chloroplasts, respectively, were detected in pumpkin leaves. Ammonium infiltrated into intact pumpkin leaves activated the synthesis of both enzyme forms. Glutamine synthetase from chloroplasts and the cytoplasmic enzyme were purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose DE-32, selective adsorption on potassium phosphate gel and preparative electrophoresis in polyacrylamide gel. The molecular weights of both forms of glutamine synthetase as determined by gel-filtration through Sephacryl S-200 are equal to 370,000 and 480,000, respectively. During SDS polyacrylamide gel electrophoresis the enzymes from both sources produced polypeptide chains with respective molecular weights of 50,000 and 58,000. The amino acid composition of the enzymes differed considerably. The content of alpha-helix moities in the chloroplast and cytoplasmic enzyme made up to 17% and 34%, respectively. In the presence of Mg+ the pH optima for the enzymes were equal to 7.75 and 7.25, respectively, and the Km values for L-glutamate were 46 and 13 mM, respectively. It may be concluded that the enzyme forms under study are isoenzymes.     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Mechanisms of regulating the activity of glutamine synthetase during ammonia assimilation]

Glutamine synthetase (GS) (E.C.6.3.1.2) activity in Chlorella cells decreased when NH4+ was added to nitrogen-free growth medium. This GS inactivation had such a rate, that it could not be due to the repression of enzyme synthesis: the GS activity decreased by 20% within 5 minutes of NH4+ assimilation. Glutamine content in cell increased in 2.5 times for this period. In vitro experiments have shown that glutamine is a strong inhibitor of GS from Chlorella grown in the presence of NO3-, and in a less degree--an inhibitor of GS from cells grown in ammonium-containing medium. The data obtained are negative with respect to possible mechanisms of GS activity regulation via adenylation and ATP-dependent destruction of glutamine synthetase.     

Fine structure of Quercus pollen from the Holocene sediments of the Sea of Japan

Holocene dispersed pollen from two cores from the shelf zone of the Korean Bay and from the deep water zone of the south of the Sea of Japan were studied by means of light and electron (scanning and transmission) microscopy. Three sculpture types were observed: rod-like, rugulate-granulate and (micro)verrucate. Ten conventional groups were separated according to the sporoderm morphology and ultrastructure. Possible specific attribution was suggested based on the comparison with published data on modern and fossil oak pollen. The perspective of further application of electron microscopy for this taxon is discussed.     

Allosteric regulators of reversible oxygenation of hemoglobin

Properties of regulators of reversible haemoglobin oxygenation, especially of a natural regulator, 2,3-diphosphoglyceric acid (DPG), are reviewed. DPG provides effective deoxygenation of haemoglobin, helps to maintain its functional properties preventing its transformation into inactive derivatives. Correlation between organism metabolic requirements and haemoglobin oxygen affinity is established by erythrocyte DPG. Several functional analogues of DPG, their structure--activity relationship and possible medical application are discussed.     

Kinetic characteristics of glutamine synthetase from the chloroplasts of pea leaves

The kinetic properties of glutamine synthetase (EC 6.3.1.2) isolated from pea chloroplasts and purified according to the previously developed procedure have been investigated. The pH optimum for the enzymatic reaction in the presence of Mg2+ and Mn2+ are 7.5-7.6 and 5.5, respectively. The corresponding values of the activation energy per enzyme monomer (Mr = 60 000) are equal to 2900 and 1190 cal/mole. With Mg2+ the values of Km(app.) for NH4+, NH2OH, L-glutamate (+NH4+), L-glutamate (+NH2OH), ATP(+NH4+ and NH2OH) and Mg-ATP (+NH4+ and NH2OH) are 0.64, 17.5, 5.6, 7.0, 1.3 and 0.74 mM, respectively.     

Secondary structure and functional groups of the active center of glutamine synthetase of pea chloroplasts

The circular dichroism spectra of glutamine synthetase (EC 6.3.1.2) from pea chloroplasts were recorded. Based on these spectra the percentage of alpha-helix sites, beta-structures, beta-bends and disordered sites of the polypeptide chain was calculated and was found equal to 23, 57, 1 and 23%, respectively. Data from protein photooxidation in the presence of methylene blue and the type of pH-dependence of pKm suggest that glutamate binding takes place on the imidazole ring of the histidine molecule. The inhibition of native glutamine synthetase by p-chloromercurybenzoate and the presence of free SH-groups in the enzyme molecule (approximately two SH-groups per monomer) suggest that these groups are the functional groups of the enzyme active center.