Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Deciduous enamel defects and caries susceptibility in a prehistoric Ohio population

Clinical studies of the relationship between developmental enamel defects and caries susceptibility have often produced conflicting results. This has been due in part to a failure to distinguish between different types of defects. Studies of this association in prehistoric populations have been rare. The complete deciduous dentitions of 57 subadults from the Libben site, a large Late Woodland cemetery in Ottawa County, Ohio, were selected for analysis. Defects were classified as either hypoplasias (deficiencies in matrix apposition) or hypocalcifications (deficiencies in mineralization) and were graded for severity. The presence or absence of carious lesions was recorded for each tooth. Results indicate a strong positive relationship between hypocalcifications and caries susceptibility. The elevated caries susceptibility of hypocalcified teeth may be related to high levels of magnesium or altered enamel microcrystallite orientation within these teeth. Variations in the frequency of hypocalcifications may partially explain differences in caries rates that have been observed in different prehistoric populations.     

Energy coupling sites in the electron transport chain of Zymomonas mobilis

Abstract Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H⁺ /O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis .     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Site-specific integration and excision of pMEA100 in Nocardia mediterranei

Summary Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.     

Subunit assembly and metabolic stability of E. coli RNA polymerase

Immunological cross-reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase generated both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, beta and beta', and the two small and intact subunits, alpha and sigma. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state. Using this method, degradation in vivo was found for some, but not all, of the amber fragments of beta subunit in merodiploid cells carrying both wild-type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.     

The Salmonella typhimurium glycine cleavage enzyme system

Summary A glycine cleavage enzyme system, inducible by glycine, has been demonstrated in Salmonella typhimurium. The induced enzyme levels, however, are only about 20% of the induced levels found in Escherichia coli. Starting with a serine auxotroph, mutants were isolated that grow with a serine supplement, but not with a glycine supplement. Three independently isolated mutants have reduced or nondetectable glycine cleavage enzyme levels. The new mutations, designated gcv, were mapped between the serA and lys genes at 62.5 min on the S. typhimurium chromosome.Abbreviations C1 one-carbon - GCV glycine cleavage - GM glucose minimal - L agar Luria agar - LB Luria broth - Tc tetracycline