Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.     

Genetic polymorphism in populations of Akodon rodents

By means of starch gel electrophoresis, 16-20 loci coding for enzymes and hemoglobin have been investigated in six population samples of Akodon dolores, captured in a single site of the Córdoba province (Argentina) during a 3-year period and in three samples of an Akodon azarae population. Proportion of polymorphic loci (P) ranged from 0.278 to 0.389 in A. dolores and from 0.166 to 0.300 in A. azarae. Mean heterozygosity (H) ranged from 0.138 to 0.192 in A. dolores and from 0.099 to 0.118 in A. azarae. These values are very high compared with those reported for northern hemisphere rodent populations. The high value is remarkable since the loci sample is biased towards the less variable (group I) enzymes.     

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Temporal variation of allele frequencies in populations of Akodon dolores (Rodentia,Cricetidae)

Summary Six population samples of the South American cricetid rodent Akodon dolores were collected at the same site at six-month intervals over a three year period. Changes in density were detected. Seven out of 18 loci analyzed by means of starch gel electrophoresis were polymorphic. Only two of these loci (Est-4 and G6pdh) showed statistically significant variation in allele frequencies following a seasonal pattern. There was no correlation between allele frequencies and population density. When animals were grouped into two classes according to body weight, a clear difference in allele distribution at the Est-4 and G6pdh loci was observed between individuals 39 g or less and those heavier than 39 g. As the first group comprises predominantly younger animals, the data indicate that changes in the age-structure of population, rather than density variations, are responsible for the cyclic pattern of allele frequencies fluctuations.     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine     

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry     

Characterization of a 3''----5'' exonuclease activity in the phage phi 29-encoded DNA polymerase.

Purified protein p2 of phage phi 29, characterized as a specific DNA polymerase involved in the initiation and elongation of phi 29 DNA replication, contains a 3'----5' exonuclease active on single-stranded DNA, but not on double-stranded DNA. No 5'----3' exonuclease activity was found. The 3'----5' exonuclease activity was shown to be associated with the DNA polymerase since 1) the two activities were heat-inactivated with identical kinetics and 2) both activities, present in purified protein p2, cosedimented in a glycerol gradient.     

Evidence for a rate-limiting role of cysteinesulfinate decarboxylase activity in taurine biosynthesis in vivo

The relationship between activities of enzymes involved in cysteine oxidation and the apparent conversion of cysteine to taurine in vivo were investigated in the rat and cat. Both hepatic cysteinesulfinate decarboxylase activity and the oxidation in vivo of cysteine to taurine were lower in the kitten than in the adult female rat and lower in the latter than in the young male rat. Our data support the hypothesis that cysteinesulfinate decarboxylase plays a rate-limiting role in taurine biosynthesis.     

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5''-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.