The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].     

Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD+, Ca2+, citrate, and isocitrate

The activity of NAD-dependent isocitrate dehydrogenase from bovine heart was inhibited by NADH (apparent Ki about 4.3 microM) and NADPH (Ki about 9.8 microM) at subsaturating substrate concentrations at pH 7.4. The inhibition by NADH or NADPH was reversed competitively by magnesium isocitrate in the presence of ADP, but not without ADP. Reversal of inhibition by NADH or NADPH with respect to NAD+ was competitive or of the linear mixed type depending on whether ADP was absent or present. ADP3- (0.2 mM) increased the Ki(app) for NADPH from 9.8 to 27.1 microM; further addition of Ca2+ (0.2 mM) raised the Ki(app) to 127 microM. For the modification of NADPH inhibition by ADP, S0.5 for Ca2+ was approximately 48 microM. This compares to the Km for Ca2+ of 0.3-1 microM for the activation of the enzyme without NADPH [Denton, R. M., Richards, D. A., & Chin, J. G. (1978) Biochem. J. 176, 899-906; Aogaichi, T., Evans, J., Gabriel, J., & Plaut, G. W. E. (1980) Arch. Biochem. Biophys. 204, 350-360]. ADP did not affect the Ki for NADH. Magnesium citrate, which was about 100-fold more effective as a positive modifier of the enzyme with ADP than without ADP [Gabriel, J. L., & Plaut, G. W. E. (1983) Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2082], reversed competitively the inhibition by NADPH in the presence of ADP, but not without ADP. Magnesium citrate did not reverse NADH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bis-(5''-guanosyl) tetraphosphatase in rat tissues.

The occurrence and distribution of bis-(5'-guanosyl) tetraphosphatase activity towards dinucleoside tetraphosphates between the 27 000 g supernatant and sedimented fraction were studied in liver, kidney, brain, muscle and intestinal mucosa from rat. The p1p4-bis-(5'-guanosyl) tetraphosphate-hydrolysing activities found in total homogenates were 0.77, 1.44, 0.39, 0.36 and 2.14 units (mumol/min)/g respectively. The activities found in the 27000 g-sedimented fractions were 74, 49, 11, 4 and 96% of those present in the homogenates respectively. The properties of the soluble enzymes were investigated. All of them have low Km values for p1p4-bis-(5'-guanosyl) tetraphosphate (from 2 to 50 microM), are competitively inhibited by guanosine 5'-tetraphosphate with K1 values from 10 to 160 nM, have molecular weights of about 21 000, require Mg2+ or Mn2+ and are inhibited by Ca2+. These properties show that bis-(5'-guanosyl) tetraphosphatase (EC 3.6.1.17), an enzyme previously characterized in Artemia salina and rat liver [Warner & Finamore (1965) Biochemistry 4, 1568-1575; Vallejo, Sillero & Sillero (1974) Biochim, Biophys. Acta 358, 117-125; Lobatón, Vallejo, Sillero & Sillero (1975) Eur. J. Biochem. 50, 495-501], is present in all the rat tissues examined. The inhibition of the enzyme by Ca2+ could be related to the effect of p1p4-bis-(5'-adenosyl) tetraphosphate as a trigger of DNA synthesis [Grummt, Waltl, Jantzen, Hamprecht, Huebscher & Kuenzle (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6081-6085].     

Membrane solubility of chlorpromazine. Hygroscopic desorption and centrifugation methods yield comparable results.

Binding of the positively charged drug chlorpromazine to artificial and erythrocyte bilayer membranes was investigated by the filtration method called hygroscopic desorption [Conrad & Singer (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202-5206] and by the conventional centrifugation method. Only minor differences in the partition coefficients were observed using the two methods. Our finding is not consistent with the observation of Conrad & Singer that amphipaths are completely excluded from biological membranes. However, the partition coefficient is dependent on membrane composition, which means dependent on the physical properties of a membrane.     

Studies on rat parotid-cell actomyosin

Actomyosin was partially purified from rat parotid cells dispersed by collagenase digestion and found to possess different solubility characteristics from that from (undispersed) rat parotid tissue. This is attributed to the decrease in vascular contamination effected by the isolation of parotid cells, yielding a non-muscle actomyosin [Adelstein, Conti, Johnson, Pastan & Pollard (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3693-3697]. Myosin light-chain kinase was partially purified from dispersed rat parotid cells by calmodulin affinity chromatography and shown to be activated by Ca2+-calmodulin. The calmodulin content of dispersed rat parotid cells was shown to be 6.50 +/- 0.59 ng of calmodulin/micrograms of rat parotid-cell protein (mean +/- S.E.M.), as determined by the activation of purified bovine brain phosphodiesterase by heat-treated extracts of dispersed rat parotid cells.     

Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings.

The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898-8902) and Asp-Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85-94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA. The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides: i + 4 AB > i + 4 BA approximately i + 3 AB > i + 3 BA. (2) The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification,classification, and analysis of beta-bulges in proteins.

A beta-bulge is a region of irregularity in a beta-sheet involving two beta-strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in beta-sheets were identified and classified automatically, extending the definition of beta-bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574-2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of beta-bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, beta-bulges produce two main changes in the structure of a beta-sheet: (1) disrupt the normal alternation of side-chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands.     

Immunoreactive insulin from mouse brain cells in culture and whole rat brain.

Foetal mouse brain cells were cultured as described previously [Sotelo, Gibbs, Gajdusek, Toh & Wurth (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 653-657] without added insulin and without foetal calf serum after 12 days in culture. Examination by phase-contrast microscopy showed that these modifications did not appear to affect growth and development of the cells adversely. Silver impregnation of the cultures and indirect immunofluorescence following reaction with tetanus toxin showed that a high proportion of the cells resembled neurones. Analysis of concentrated culture medium by radioimmunoassay and high-pressure liquid chromatography (h.p.l.c.) revealed that the cells produced two main forms of immunoreactive insulin which differed from authentic pancreatic insulin in retention time. Immunoreactive somatostatin was also produced in culture and this was resolved into at least three forms by h.p.l.c. Immunoreactive insulin was also extracted from whole rat brain by using two published procedures. The method of Havrankova, Schmechel, Roth & Brownstein [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 5737-5741] consistently gave greater yields of insulin than did that of Eng & Yalow [Diabetes (1980) 29, 105-109] and the concentration was about three times that of plasma. The extracted insulin was further characterized by h.p.l.c. in each case and was found to behave like authentic pancreatic insulin. The production of insulin and somatostatin by foetal mouse brain cells in culture suggests that they may be a useful model system for studies of neuropeptide biosynthesis.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence.

Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.     

Physical studies on glycoproteins from ascitic fluid of ehrlich ascites tumor

Three glycoproteins, designated as F, M and S glycoproteins were identified in the HClO₄-soluble fraction of ascitic fluid of Ehrlich ascites tumor by 8% polyacrylamide disc gel electrophoresis. They were separated and purified as described previously (Reznick, A.Z. and Winzler, R.J. (1973) Fed. Proc. 32, 368 and Reznick, A.Z., Allen, H.J. and Winzler, R.J. (1973) Anal. Biochem. 52, 395–401) and subjected to physical characterization. Several physical properties such as molecular weights, sedimentation and diffusion coefficients, partial specific volumes, Stoke's radii and frictional ratios were determined. The physical parameters of F and S glycoproteins resemble data that have been reported for orosomucoid and haptoglobin-like glycoproteins, respectively. Properties of M glycoprotein could not be associated with a known glycoprotei.     

Protein degradation in rat liver during post-natal development.

Protein degradation rates for liver subcellular and submitochondrial fractions from neonatal (8-day), weanling (25-day) and adult rats were estimated by the double-isotope method with NaH14CO3 and [3H] arginine as the radiolabelled precursors [Dice, Walker, Byrne & Cardiel (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097]. Decreased protein degradation rates were found during post-natal development for homogenate, nuclear, mitochondrial, lysosomal and microsomal proteins. A decrease in degradation rates for the immunoisolated subunits of monoamine oxidase and pyruvate dehydrogenase was also observed in neonatal and weanling rats respectively. The results suggest coordinate degradation of the subunits of the multi-subunit enzyme pyruvate dehydrogenase. Pyruvate dehydrogenase has a faster rate of degradation in adult rat liver than does cytochrome oxidase. Data analysis suggests heterogeneity of protein degradation rates in the mitochondrial outer membrane and intermembrane space fractions at each developmental stage but not in the mitochondrial inner membrane or matrix fractions. Results obtained for protein degradation rates in adult rat liver by the method of Burgess, Walker & Mayer [(1978) Biochem. J. 176, 919-926] in general confirmed the results obtained for the adult rat liver by the above method. No evidence of a subunit-size relationship for protein degradation was found for proteins in any subcellular or submitochondrial fraction.     

A novel method for the synthesis of nucleoside triphosphates labelled with inorganic [32P]phosphate specifically in the beta-position.

A method was developed for the introduction of [32p]Pi specifically into the beta-position of ATP and GTP. The method is based on two separate reactions involving (a) phosphorolysis of poly(A) or poly(G) [Soreq, Nudel, Salomon, Revel & Littauer (1974) J. Mol Biol. 88, 233-245] in the presence of [32P]Pi and (b) conversion of the labelled diphosphate into the corresponding triphosphate by transferring the active phosphate group from 1,3-diphosphoglycerate in a coupled reaction as decribed by Glynn & Chappell [(1964) Biochem. J. 90, 147-149]. Radioactivity in the beta- and gamma-phosphate groups of the labelled triphosphate was measured by using polynucleotide kinase. No detectable radioactivity was found in the gamma-phosphate group. The suitability of this method for the synthesis of other nucleoside triphosphates specifically labelled in the beta-position is discussed.     

Comparison of Compatible and Incompatible Response of Potato to Meloidogyne incognita

One susceptible (D6) and two resistant (E2 and N4) clones of Solanum sparsipilum × (S. phureja × haploid of S. tuberosum) were used to study the responses of potato roots and tubers to race 1 of Meloidogyne incognita (Kofoid &White) Chitwood. The compatible response was characterized by rapid penetration of large numbers of second-stage juveniles (J2) into roots, cessation of root growth, and occasional curving of root tips. The life cycle of M. incognita in the susceptible clone was completed in 25 days at 23-28 C. The incompatible response was characterized by penetration of fewer J2 into roots, necrosis of feeding sites within 2-7 days, and lack of nematode development. There were no differences in response of tubers from resistant and susceptible clones to nematode infection. Small numbers of J2 were detected in tubers, but they did not develop.     

Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53.

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.     

The relationship of the rat brain 68 kDa microtubule-associated protein with synaptosomal plasma membranes and with the Drosophila 70 kDa heat-shock protein.

A protein of molecular mass 68 kDa and pI5.6 is a major translation product of rat brain mRNA [Hall, Mahadevan, Whatley, Biswas & Lim (1984) Biochem. J. 219, 751-761]. In the rat brain this protein was associated with microtubule preparations and was present together with tubulin as a component of the synaptosomal plasma membranes, synaptic vesicles and post-synaptic structures. The brain mRNA for this protein was found to hybridize specifically to the Drosophila gene for the 70 kDa heat-shock protein, thus enabling its rapid isolation.     

Different reactivities of free and bound amino groups in deoxy-and liganded haemoglobin.

The technique of competitive double-labelling [H. Kaplan, K.J. Stevenson & B.S. Hartley, (1971) Biochem. J. 124, 289-299; L.P. Visentin & H. Kaplan (1975) Biochemistry 14, 463-468] was used to determine the reactivity of some amino groups towards acetic anhydride in deoxy-and liganded haemoglobin. Only those amino groups known to form salt bridges in deoxy-but not in liganded haemoglobin (i.e. the alpha-amino group of valine-1 alpha and the xi-amino group of lysine-40 alpha and lysine-127 alpha [M. F. Perutz (1970) Nature (London) 228, 726-739]) and different reactivities in the two structures.     

Channeling of extramitochondrial ornithine to matrix ornithine transcarbamylase

In the presence of citrulline synthesis, we made the following observations. External ornithine is channeled between its transporter and ornithine transcarbamylase; mitochondria preloaded with cold ornithine, then incubated with [3H]ornithine, produced citrulline of the same specific radioactivity as that of external ornithine, while matrix ornithine remained essentially unlabeled. The channeling of ornithine suggests that some soluble enzymes are organized within the mitochondrial matrix. The rate of ornithine transport can be greater than 80 nmol/min/mg. At rates of carbamyl phosphate synthesis of 10-50 nmol/min/mg, the rate of citrulline synthesis is controlled by external ornithine in the range 0.03-0.2 mM; at greater than or equal to 0.2 mM ornithine, transport is not limiting for citrulline synthesis. At external ornithine concentrations less than or equal to 1 mM, i.e. within the physiological range, this amino acid is undetectable in the matrix. Given the rates of citrulline and urea synthesis which occur in vivo and the concentrations of ornithine present in the liver, our findings indicate that ornithine may contribute to the physiological regulation of urea synthesis. Preliminary reports of parts of this work have been published (Raijman, L., Cheung, C-W., and Cohen, N. S. (1984) Fed. Proc. 43, 1831; Cohen, N. S., Cheung, C-W., and Raijman, L. (1986) Fed. Proc. 45, 2677).     

Purification and properties of lung lectin. Rat lung and human lung beta-galactoside-binding proteins.

Lung is one of the organs of the rat with a particular abundance of haemagglutinating activity that is inhibited by beta-galactosides. This lectin activity can be attributed to a single protein that has been purified from rat lung; a similar protein has been purified from human lung. The molecular weights and subunit structures were estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the human lung lectin appeared to be composed to two identical subunits, mol.wt. 14500, whereas rat lung lectin was observed as both a dimer and a tetramer of one subunit type, mol.wt. 13500. Both lectins bind to disaccharides or oligosaccharides with terminal beta-linked galactose residues. The carbohydrate moiety may be free [lactose or D-galactopyranosyl-beta-(1 leads to 4)-thiogalactopyranoside], protein-bound (asialofetuin) or lipid-bound (cerebrosides). The molecular properties of the beta-galactoside-binding proteins of rat lung and human lung are closely similar to those of embryonic chick muscle lectin [Nowak, Kobiler, Roel & Barondes (1977) Proc. Natl. Acad. Sci. U.S.A. 73, 1383--1387] and calf heart lectin [De Waard, Hickman & Kornfeld (1976) J. Biol. Chem. 251, 7581--7587].     

Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.     

Synthesis of High Titer Infectious Prions with Cofactor Molecules

Recently, synthetic prions with a high level of specific infectivity have been produced from chemically defined components in vitro. A major insight arising from these studies is that various classes of host-encoded cofactor molecules such as phosphatidylethanolamine and RNA molecules are required to form and maintain the specific conformation of infectious prions. Synthetic mouse prions formed with phosphatidylethanolamine exhibit levels of specific infectivity ∼1 million-fold greater than “protein-only” prions (Deleault, N. R., Walsh, D. J., Piro, J. R., Wang, F., Wang, X., Ma, J., Rees, J. R., and Supattapone, S. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E1938–E1946). Moreover, cofactor molecules also appear to regulate prion strain properties by limiting the potential conformations of the prion protein (see Deleault et al. above). The production of fully infectious synthetic prions provides new opportunities to study the mechanism of prion infectivity directly by structural and biochemical methods.