A suppressor analysis of residues involved in cation transport in the lactose permease: identification of a coupling sensor.

Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the alpha-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and DeltaI298). All of the suppressors transported alpha-galactosides at substantial rates. In addition, the Q60L, DeltaI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H(+) or H(3)O(+)). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.

Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli.

Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.

Growth of native Hawaiian school children: II. Body mass index and skinfold measurements.

Annual measurements of skinfold thicknesses at six sites (triceps, biceps, medial calf, subscapular, abdominal, and suprailiac) and the body mass index have been obtained from a sample of native Hawaiian school children and their non-Hawaiian classmates in a four year semilongitudinal study. Four cohorts were measured, with the children beginning the study in grades one, four, seven and ten, respectively. Information on percentage of Polynesian ancestry, identity with Hawaiian culture, social class, and income were obtained from family interviews for the native Hawaiian children. Hawaiian boys at most ages surveyed are significantly fatter than their classmates on trunk sites, and are also fatter than National Health and Nutrition Examination Survey II (NHANES-II) medians. Native Hawaiian girls have significantly greater trunk skinfold measures than classmates in only one surveyed age group. The percentage of Polynesian ancestry in this admixed sample of Hawaiian children is significantly related to fatness and body mass among children aged 9-12 years, but is not clearly related to fatness in other age groups. Socioeconomic measures are also not related to fatness in a simple manner. Intensive study of specific sex-age groups may be required to identify factors that influence the amount of fatness of native Hawaiian school children. © 1992 Wiley-Liss, Inc.

Growth of native Hawaiian school children: I. Research design, stature, and weight measurements.

A semilongitudinal survey of the growth of native Hawaiian school children, focused on the development of fatness, has been carried out on the Island of Hawaii. Four cohorts of children, first studied in grades one, four, seven, and ten, were measured annually for four years. Several factors, including percentage of Polynesian ancestry, identification with Hawaiian culture, socioeconomic status, dietary intake, and physical fitness, have been considered for their potential relationship to the development of fatness Data presented here indicate that native Hawaiian boys tend to be taller for their age than NHANES-II medians until age 14 years, but are not significantly taller than their non-Hawaiian classmates. Native Hawaiian boys and girls are heavier for their age than NHANES-II medians at most ages and are significantly heavier than non-Hawaiian classmates. Percentage of Polynesian ancestry is a significant predictor variable of both stature-for-age and weight-for-age among native Hawaiian children in the first and fourth grade cohorts. Study of fatness in a genetically heterogeneous Polynesian population that is relatively homogeneous in regard to modernization may yield insights into genetic contributions to the problem of obesity among Pacific Islanders.