Methionyl-tRNA synthetase of Escherichia coli. A zinc metalloprotein.

The native dimeric form of methionyl-tRNA synthetase of Escherichia coli contains two zinc atoms per dimer, one per subunit. The bound zinc is retained upon trypsin modification which yields a monomer with one zinc atom. The enzymatic activity of both the dimeric forms is reversibly inhibited by 1,10-phenanthroline but not by its non-chelating analogues. In addition, the native enzyme binds two Mn2+ per dimer with a binding constant of approx. 70 micron but no binding is observed with the trypsin-modified monomer.     

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.     

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.     

OCCURRENCE OF A STELAR LESION DURING IMBIBITIONAL CHILLING OF ZEA MAYS L.

Radicle growth of the corn inbred Oh51A was reduced by imbibitional chilling at 5 C. The effect was mediated by the initial moisture of the kernel prior to hydration. Five percent initial moisture kernels were injured while 13% initial moisture kernels were not. The formation of a structural lesion in the radicle during the first 24 hr of hydration at 5 C of 5% initial moisture kernels was correlated with subsequent radicle growth reduction at 25 C. The lesion did not form during hydration of 13% initial moisture kernels at 5 C, in kernels hydrated at 25 C, in unimbibed kernels, or in heat-killed, cold-imbibed kernels. The lesion also did not appear during the imbibitional chilling of the corn inbred B8 which did not exhibit radicle growth reduction. Two sources of Oh51A with similar lesion frequencies were different in the severity of growth reduction. While the lesion became sealed at 25 C subsequent to cold hydration, growth reduction differences were not fully explained by this phenomenon. In one source more seedlings exhibited reduced growth than expected from the frequency of observable lesions. Therefore, other types of damage contributed to the reduction of radicle growth.     

31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding.

The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Calcium-binding protein of the chick chorioallantoic membrane. I. Immunohistochemical localization

The preparation of a specific antiserum (anti-CaBP) against the calcium-binding protein (CaBP) of the chorioallantoic membrane (CAM) is described. The anti-CaBP appeared to be specific for the CaBP by immunodiffusion and immunoelectrophoresis. Application of the anti-CaBP in immunofluorescence histochemistry revealed that the CaBP is present in the CAM only at developmental ages corresponding with the expression of the calcium transport function of the membrane. Furthermore, the CaBP is localized to the ectoderm of the CAM, appears to be exposed to the entire external surface of the ectoderm, and can be shown to be associated with cells enzymatically dissociated from the CAM. These results are consistent with a functional role of the CaBP in the CAM calcium transport process.     

Seed Dormancy in Red Rice : VIII. Embryo Acidification during Dormancy-Breaking and Subsequent Germination

Exposure of dehulled, dormant red rice (Oryza sativa) seeds to dormancy-breaking treatments (10 mm sodium nitrite, 20 mm propionic acid, 30 mm methyl propionate, 40 mm propionaldehyde, or 70 mmn-propanol) induced tissue pH acidification during chemical contact at least 12 h before visible germination. During chemical contact, the onset of embryo acidification occurred before or coincident with the chemical contact interval necessary for subsequent germination. Upon seed transfer to H₂O following chemical contact, embryo pH also decreased coincident with visible germination. During this period, the percentage of germination and embryo pH were closely linked irrespective of the dormancy-breaking compound used. Therefore, tissue acidification during the breaking of seed dormancy and the germination process may be analogous to similar tissue pH changes associated with the termination of developmental arrest in other multicellular systems, such as brine shrimp cysts and nematode larvae.     

Intralysosomal accumulation of polyanions. II. Polyanion internalization and its influence on lysosomal pH and membrane fluidity

Dextran sulfate (DS) was previously shown to inhibit phagosome-lysosome (P-L) fusion whereas dextran (D) of equivalent size was ineffective. The uptake and interiorization of DS were examined with a tritiated product over the course of 4 d in culture. The exposure of macrophages to 20 micrograms/ml of 3H-DS led to linear uptake for 4 d, at which time fusion was inhibited. Macrophage interiorization of 3H-DS was greatly increased by forming insoluble complexes with either serum lipoproteins or purified human low density lipoproteins (LDL). Under these conditions fusion was inhibited within 4 h. The uptake of large quantities of acetylated LDL in the absence of DS was not associated with the inhibition of fusion. Lipoproteins therefore served as the DS carriers and were not themselves inhibitory. The intralysosomal pH of control and D-treated macrophages was 4.76 (+/-0.06) and 4.68 (+/-0.02), respectively. Storage of DS was associated with a decreased pH to 4.36 (+/-0.14). Increasing the intralysosomal pH with either NH4Cl or chloroquine failed to modify inhibited P-L fusion. Hydrogen ion concentration was therefore not an important factor in DS inhibition. Secondary lysosomes were isolated from D- and DS-loaded cells and exhibited excellent latency. These lysosomes were exposed to the membrane probes, alpha- and Beta-parinaric acid, and compared in fluorescence polarization measurements. The results with the Beta isomer consistently indicated that the membranes of DS lysosomes were more rigid than the D samples. It is suggested that high intralysosomal concentrations of DS interact directly with either lipid and/or polypeptide moieties of the luminal face of the membrane, thereby decreasing its fluidity and fusibility.