Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Isolation and sequence of a peptide containing an essential lysine

Interaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with pyridoxal 5'-phosphate and sodium borohydride leads to inactivation and modification of two lysine residues per enzyme dimer that are thought to bind glucose 6-phosphate [Milhausen, M., & Levy, H.R. (1975) Eur. J. Biochem. 50, 453-461]. The amino acid sequence surrounding this lysine residue is reported. Following tryptic hydrolysis of the modified enzyme, two peptides, each containing one pyridoxyllysine residue, were purified to homogeneity and subjected to automated Edman degradation. The sequences revealed that one of these, a heptapeptide, was derived from the other, containing 11 amino acids. Supporting evidence for the role of the modified lysine is provided in the following paper [Haghighi, B., & Levy, H.R. (1982) Biochemistry (second paper of three in this issue)]. End-group analysis of the native enzyme revealed that valine is the N-terminal and glycine the C-terminal amino acid and provides support for the identity of the enzyme's two subunits.     

marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.     

Location-dependent variations in the material properties of the anterior cruciate ligament.

Our recent anterior drawer studies in human cadaveric knees [Guan and Butler, Adv. Bioengng 17, 5 (1990); Guan et al., Trans. orthop. Res. Soc. 16, 589 (1991)] have suggested that anterior bundles of the anterior cruciate ligament (ACL) develop higher load-related material properties than posterior bundles. This was confirmed when we reevaluated the axial failure data for these bundle-bone specimens from an earlier study [Butler et al., J. Biomechanics 19, 425-432 (1986)]. The purpose of this study was to determine, in a larger data set, if anteromedial and anterolateral bundles of the anterior cruciate ligament exhibit significantly larger load-related material properties than the posterior ligament bundles. Seven ACL-bone units from seven donors (the three tissues from the original study plus four new ones) were subdivided into three subunits, preserving the bone insertions. The subunits were failed in tension at a constant strain rate (100% s-1) and four material properties were compared within and between donors. The anterior bundles developed significantly larger moduli, maximum stresses, and strain energy densities to maximum stress than the posterior subunits. Moduli for the anterior vs posterior subunits averaged 284 MPa vs 155 MPa, maximum stresses averaged 38 MPa vs 15 MPa, and strain energy densities averaged 2.7 N m cc-1 vs 1.1 N m cc-1, respectively. No significant differences were found, however, among strains to maximum stress or between any of the other properties for the two anterior subunits. These results are important to the design of ligament replacements and suggest new experiments designed to distinguish in vivo force levels in these ACL bands, a possible reason for the material differences.     

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.     

The 1991 Borden Award Lecture. Selected aspects of intraluminal and intracellular phases of intestinal fat absorption.

The recognition of chylomicrons as dietary lipid transporters dates back to more than 70 years and marks a milestone in lipoprotein history. Conventionally, three phases constitute the process of absorption of exogenous fat: intraluminal, intestinal, and delivery. The intraluminal phase includes chemical hydrolysis by lipolytic enzymes and the micellar solubilization of lipolytic products by bile acids. The intestinal phase comprises the diffusion of micelles through the unstirred water layer, passive diffusion across the microvillous membrane of the enterocyte, and the formation of lipid-carrying lipoproteins. The delivery phase involves the exocytosis of chylomicrons from the absorptive cells and their subsequent removal by lymphatic structures and the systemic circulation. The precise steps and factors involved in all phases of chylomicron synthesis are not yet known, but both experimental and clinical studies have been helpful. Of the inborn metabolic disorders, the prerequisite function of apolipoprotein (apo B) for the assembly and release of lipoprotein particles stood out. Moreover, evidence emerged that the enterocyte produces apo B-100 in addition to apo B-48. Calcium and essential fatty acid status originates as determinants for triglyceride-rich particle synthesis. Furthermore, the developmental changes and regulatory factors of lipoprotein elaboration represent excellent tools in the study of the intracellular mechanisms of lipid transport.     

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.     

Disease Development and Buildup of Resistance to Oxadixyl in Potato Crops Inoculated with Phytophthora infestans as Affected by Oxadixyl and Oxadixyl Mixtures: Experimental and Simulation Studies

Simulator predictions for the effect of oxadixyl and oxadixyl mixtures on the control of late blight in potato crops inoculated with Phytophthora infestans and the buildup of fungal resistance to oxadixyl were compared with data obtained in field experiments. The simulator, which considered fungus, fungicides and crop variables, predicted accurately the effect of fungicide sprays (oxadixyl, oxadixyl mixed with mancozeb, and oxadixyl mixed with mancozeb and cymoxanil) on blight development. The model predicted accurately both resistance buildup and infection in non-treated crops and in crops treated with oxadixyl alone or in a mixture with mancozeb (SAN 518F), but overestimated the control efficacy of the three-way mixture Pulsan.