In vitro renin inhibition to prevent generation of angiotensins during determination of angiotensin I and II

To prevent in vitro generation of angiotensins, the renin inhibitor CGP 29287 (CGP) was added to blood sampling tubes. Plasma immunoreactive angiotensin (ir-ANG) I and II were simultaneously measured by radioimmunoassay after rapid and quantitative extraction from a single plasma sample on phenylsilylsilica (Bondelut PH). True plasma ANG-(1-8)octapeptide was determined after additional separation of the different angiotensins by high performance liquid chromatography. Ir-ANG II/CGP showed the known linear relationship with ANG-(1-8)octapeptide (r = 0.87, n = 23), but - in contrast to studies without addition of CGP - the y-axis intercept which presumably represents cross-reacting angiotensins other than ANG II was very small. Ir-ANG II/CGP concentrations fell below 1 fmol/ml after converting enzyme inhibition. The results suggest that CGP 29287 prevents in vitro generation of ANG I and ANG II as well as the ANG-metabolites. Ir-ANG I/CGP measured after Bondelut PH extraction of the plasma was strongly correlated with ir-ANG I obtained after blood ethanol extraction (r = 0.97, n = 23). Thus, it is now possible to measure reliably both ANG I and ANG II within the same plasma extract after a simple extraction procedure.     

Deaggregation and proteolytic modification of a galactosyltransferase of Poterioochromonas malhamensis

We isolated hybridomas that produced monoclonal antibodies specific for the UDP-galactose: sn -glycerol-3-phosphate α-D-galactosyltransferase (IFP synthase, EC 2.4.1.96), an enzyme involved in the volume regulation of Poterioochromonas malhamensis Peterfi. Western blotting of native gradient gels with the most reactive antibody S 162 revealed several immunoreactive proteins in crude homogenates suggesting the occurrence of multiple molecular mass species of the galactosyltransferase. The amount of the presumed enzyme monomer (64 kDa under native conditions) was strongly increased by a pH shift of crude homogenates from pH 8 to 6. During activation of the galactosyltransferase in the cell homogenate and also by shrinking the cells, the presumed enzyme monomer appeared to be proteolytically degraded generating stepwise products of 52 and 40 kDa. We assume that the proteolytically processed enzyme becomes highly active, but is very susceptible to further proteolytic degradation.     

The role of tungstate and/or molybdate in the formation of aldehyde oxidoreductase in Clostridium thermoaceticum and other acetogens; immunological distances of such enzymes

Besides Clostridium thermoaceticum and C. formicoaceticum other resting acetogenic clostridia such as C. aceticum and C. thermoautotrophicum and to a lesser extent non-clostridial acetogens such as Butyribacterium methylotrophicum and Eubacterium limosum were able to reduce propionate to propanol at the expense of carbon monoxide or formate. Methylviologen usually increased the reduction rate. Ten M molybdate in the growth medium decreased this capability for C. thermoaceticum but increased it or had no effect for the other organisms. Ten M tungstate in the growth medium increased the aldehyde oxidoreductase activity in all organisms. Crude extracts of C. thermoaceticum cells grown in the presence of 10 M or 1 mM molybdate showed by ELISA the same or even a 4 fold concentration of aldehyde oxidoreductase in the latter case. However, the enzymic activity was very low in both cases. Omission of dithionite in the growth medium diminished the antigen by a factor of about 8. The immunological distance between the enzyme from C. thermoaceticum and C. thermoautotrophicum was rather low but very large to C. formicoaceticum and undeterminably large to the other organisms.Abbreviations Ald-DH aldehyde dehydrogenase - AOR aldehyde oxidoreductase - CO-DH carbon-monoxide dehydrogenase - ELI-SA enzyme-linked immunosorbent assay - FDH formate dehydrogenase - MV methylviologen - V⁺⁺ oxidised - V^+. reduced viologen     

Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene

A 2.4 kb fragment containing the 5'-flanking region and the 5'-noncoding sequence of the Vicia faba legumin gene LeB4 mediates high level seed-specific expression in transgenic tobacco plants. Deleted derivatives of this legumin upstream sequence were fused to the npt-II reporter gene to determine the tissue-specific activity of the chimeric constructs in stably transformed tobacco plants. The results indicate the presence of positive regulatory, enhancer-like cis elements within 566 bp of the upstream sequence. Most importantly, however, these elements are only fully functional in conjunction with the core motif CATGCATG of the legumin box around position -95, since destruction of the motif by a 6 bp deletion in an otherwise intact 2.4 kb upstream sequence drastically reduces expression in seeds. At the same time, low level expression in leaves is observed. The occurrence of similar CATGCATG consensus cis elements with alternating purine and pyrimidine base pairs in front of several other plant genes suggests a functional role of the motif in a wider range of plant promoters.     

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.     

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.     

Developmental modulation of a glial cell-associated glycoprotein, 5B12, in an insect, Acheta domesticus

The expression of an insect (Acheta domesticus) adult glial cell-specific antigen, 5B12 undergoes major changes during development. The 5B12 antigen is detected as early as 20-25% of embryonic development, when immunoreactivity is distributed throughout the periphery, present at the luminal surface of epithelial cells which compose developing limb buds, sensory appendages, and the body cavity. The antigen is also localized on the cell surface of neural elements within commissural tracts in the embryonic CNS. 5B12 is secreted extracellularly in the periphery, where it is associated with the embryonic basal lamina in developing cercal sensory appendages. Luminal surface expression is transient, and disappears by 95% of embryonic development. As development proceeds, 5B12 distribution becomes more restricted, so that in the adult the antigen is predominantly associated with specific glial elements within the nervous system where it occurs as a specialized component of the extracellular matrix. The 5B12 antigen is also associated with discrete central and peripheral fiber tracts. Antigen 5B12 is present in whole embryos and in the adult CNS as a Mr 185-kDa glycoprotein. Distinct carbohydrate moieties with chondroitin sulfate-like properties are situated on the 5B12 epitope. Thus the glia-associated 5B12 macromolecule has the characteristics of a small proteoglycan. Based upon features of its distribution, pattern of spatiotemporal expression, and biochemical properties, it is speculated that 5B12 participates in events related sequentially to the development and the function of the insect nervous system.     

Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.     

Neuropeptide Y normalizes renin secretion in adrenalectomized rats without changing blood pressure

In the periphery, neuropeptide Y is present in plasma, in the adrenal medulla as well as in sympathetic nerve endings and in the juxtaglomerular apparatus. The aim of the present study was to assess the effect of this peptide on renin secretion. Normotensive rats were adrenalectomized or sham-operated and made hypertensive with methylprednisolone acetate (20 mg/kg s.c. once weekly). Deoxycorticosterone pivalate (10 mg/kg s.c. once weekly) was also given to prevent mineralocorticoid deficiency. Two weeks after initial surgery, 12 adrenalectomized and 8 sham-operated conscious rats were infused for 30 min with neuropeptide Y (0.1 micrograms/min) whereas 8 other adrenalectomized and 9 sham-operated conscious rats received under similar conditions the vehicle of neuropeptide Y (10 microliter/min). Neither before nor during the infusions was there a significant difference in blood pressure and heart rate between the 4 groups of animals. Plasma renin activity, measured at the end of the infusion, was 30.5 ng/ml/hr in the adrenalectomized group receiving vehicle and 6.3 ng/ml/hr in that infused with neuropeptide Y (p less than 0.001). This latter value did not differ from that found in sham-operated rats. These results suggest that neuropeptide Y may play an important role in regulating renin secretion.