Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.     

Conversion of dihomo-gamma-linolenic acid to mono- and dihydroxy acids by potato lipoxygenase: evidence for the formation of 8,9-leukotriene A3

Evidence for the formation of a positional isomer of leukotriene (LT) C3 (8,9-LTC3) from dihomo-gamma-linolenic acid has been published (Hammarstr?m, S. J. Biol. Chem. 256, 7712-7714, 1981). This report describes the conversion of dihomo-gamma-linolenic acid to a postulated intermediate in former reaction, 8,9-LTA3, by purified lipoxygenase from potato tubers. 8(S)-Hydroperoxyeicosatrienoic acid (8(S)-HPETrE) was the most abundant dioxygenation product formed followed by 11-, 15-, and 12-HPETrEs (in decreasing order of abundance). In addition, 8(S),15(S)- plus 8(S), 15(R)-dihydroperoxyeicosatetraenoic acid (DiHPE-TrE) (EZE), and 8(S),15(S)- plus 8(S),15(R)-dihydroxy-eicosatetraenoic acid (DiHETrE) (EEE) were generated. Under anaerobic conditions only the latter two isomers of 8,15-DiHETrE (EEE) were obtained from 8-HPETrE. The results suggest that 8,9-LTA3 is synthesized by the sequential action of 8- and 11-lipoxygenase activities associated with the potato enzyme.     

Specific antibodies and mycobacterial antigens in pulmonary tuberculosis patients sera. Note I

206 sera collected from different groups of subjects were analyzed by immunoenzymatic methods regarding the content of Mycobacterium tuberculosis specific antibodies and mycobacterial antigens. The results underlined that two assays offered improved serologic diagnosis of tuberculosis over a single antibody test. BCG vaccination interferes with serologic tests for tuberculosis when polyspecific antibodies or mixture of common and specific antigens are used as immunologic reagents. A mycobacterial antigens circadian variation in correlation with vesperal fever in tuberculous patients was not revealed. The mycobacterial antigens seem to become undetectable, in sera, after 6 months of efficient treatment.     

Microcirculation, hemodynamics and blood rheology in peritonitis during plasmapheresis

By means of biomicroscopy, electrophysiological and hemorheological techniques effect of plasmapheresis with use of various plasma -substituting solutions and their combination on microcirculation, hemodynamics and rheology of blood at peritonitis has been studied in the experiment on 329 white non-inbred rats and on 50 mature dogs. Three periods of disturbances in hemomicrocirculation of the small intestine mesentery at peritonitis have been revealed: I (3-6h)--a relative well-being and compensatory reactions in the system of hemomicrocirculation; II (9-15h)--development of disturbances in the aggregate state of blood; III (18-24h)--pronounced disturbances in hemomicrocirculation. Plasmapheresis at peritonitis facilitates: to better functioning of the microcirculatory system, namely, a sharp increasing of the blood flow rate, joining into the blood flow of capillaries that did not function previously and increasing functional capacity of the capillary bed; decreasing blood viscosity and aggregation of erythrocytes. Plasmapheresis applied at peritonitis produces a favourable effect to hemodynamics, evident as an increasing minute and stroke volume of the blood flow, cardiac index, the left ventricle activity and decrease in peripheral vascular resistance.     

Identification of a new carcinoembryonic antigen (CEA) family member in human fetal liver--cloning and sequence determination of pregnancy-specific glycoprotein 7

The carcinoembryonic antigen gene family consists of the CEA- and the Pregnancy-Specific Glycoprotein- (PSG) subfamilies. Human fetal liver express several PSGs. Here we report cloning and sequencing of a new PSG subfamily member, PSG7. It is the fifth type of PSG found in fetal liver. PSG7 has the N-A1-A2-B2-C domain arrangement. Unlike other PSGs the N-terminal of PSG7 is unblocked. PSG7 has a cysteine in the C-terminal domain, which may allow dimerization. Variability analysis according to Wu and Kabat reveals that the region in the N-domain corresponding to complementarity determining region 3 of immunoglobulin is different between PSG subfamily members. Many members, including PSG7, contain the RGD sequence in this region. The CD2 region as well as two other short sequences (in N and A1 domains respectively) also show some variability. The function of PSGs is probably linked to the N-domain and the CDR2- and CD3-like regions are most likely responsible for ligand binding.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.     

Cis-trans isomerization of an angiotensin I-converting enzyme inhibitor. An enzyme kinetic and nuclear magnetic resonance study

The angiotensin I-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) inhibitor, ramiprilat (2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-Ala]-(1S,3S,5S)-2- azabicyclo[3.3.0]octane-3-carboxylic acid), is shown to exist in tow conformational isomers, cis and trans, which interconvert around the amide bond. The two conformers were separated by reversed-phase high-performance liquid chromatography. The conformers were identified by nuclear Overhauser effect measurements. From line shape analysis the isomerization rate constants were determined to be kcis----trans = 15 s-1 and ktrans----cis = 5 s-1 at 368 K in [2H]phosphate buffer (p2H 7.5). By enzyme kinetic studies using 3-(2-furylacryloyl)-L-Phe-Gly-Gly as substrate, the trans conformer was found to be the most potent enzyme inhibitor, whereas the cis conformer had a very low inhibitory effect. A new inhibition mechanism is presented for this type of slow, tight-binding inhibitors that contain an amide bond. This mechanism involves an equilibrium between the two conformers and the enzyme-bound inhibitor complex.     

Stimulation of ATP-driven Ca2+ pump in the basal-lateral plasma membranes of kidney cortex during compensatory renal growth

During compensatory renal growth 45Ca2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca2(+)-ATPase activity was observed, without an effect of compensatory renal growth on Na+/Ca2+ exchanger activity and on passive Ca2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca2(+)-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca2(+)-ATPase were studied in native membranes, an increase of Vmax was observed, whereas the Km for Ca2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of Vmax and an increase of Km (Ca2+), while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of Vmax in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca2(+)-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.