Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli

The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli . The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the Α subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK , near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the Α-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38450/1/240460407_ftp.pd

The substrate-facilitated transport of the glucose carrier across the human erythrocyte membrane

1. 1. The rate of egress of glucose from human red blood cells into saline media either without sugar or containing glucose, galactose, sorbose or fructose has been determined at 25[deg] and for glucose or zero sugar at 0[deg].2. 2. From these data, using a simplified form of the theoretical analysis of 13, it is shown that the rate of movement across the membrane of the substrate-carrier complex is greater than that of the free carrier, that is, the movement of carrier is facilitated by the substrate.3. 3. A lower limit of 2.8 is derived for the ratio of these rates of movement.4. 4. Evidence is presented that the aldoses and ketoses share a common carrier.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31976/1/0000018.pd

Regulation of amino acid transport system L by amino acid availability in CHO-K1 cells. A special role for leucine

Starvation of CHO-K1 cells for leucine leads to a 3-4-fold increase in transport system L activity, without modification of transport through systems A and ASC. The concentration of leucine must be below 10 [mu]M before the enhancement of transport can be clearly seen. To achieve low concentrations of leucine such as 10 [mu]M, extensive dialysis of fetal calf serum was required. The enhancement of transport was completed after 12-24 h of starvation and was fully reversed within 1 h of re-feeding with leucine. Starvation for isoleucine, valine or phenylalanine also produced an increase in system L transport activity, but the effect was only one half of that seen following leucine starvation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25535/1/0000076.pd

On the meaning of effects of substrate structure on biological transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44802/1/10863_2005_Article_BF01516050.pd

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn -glycerol-3-phosphate transport system in Escherichia coli

The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn -glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR -dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho -independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho -independent ugp expression correlated with the presence of the cya and crp gene products.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47580/1/438_2004_Article_BF00290646.pd

Leucine and tissue distribution of bulky and small neutral amino acids in rats: Dissociation between transport and insulin-mediated effects

The mechanism of the observed decrease in the plasma concentration of several amino acids in the presence of high levels of Leu has remained unexplained. In the present study a decrease in the plasma concentration of Ile, Val, Phe, Tyr, Met, Ala, Pro and Gly was observed after the intraperitoneal injection of Leu to weanling rats. Decreases in net intracellular concentrations in muscle accompanied the decrease in plasma of all of these amino acids except Pro and Gly. An increase in the distribution ratio muscle/plasma was observed exclusively for Gly after administration of Leu or of a non-insulinogenic transport system L analogue. Diazoxide suppressed the Leu-induced decreases in plasma and muscle intracellular concentrations of Ile and Val as well as of Pro in plasma. An increase in the distribution ratio liver/plasma was observed for Pro and Gly in the absence but not in the presence of diazoxide. All the above changes were statistically significant. Hence insulin probably mediates Leu effects, promoting an increased utilization of Ile and Val in muscle and of Pro in liver. A more direct effect of Leu appears to be involved in the apparent increased utilization of Phe, Tyr and Ala in the same tissue. Gly depletion in plasma can be explained by its trapping by inhibitory action of Leu on the exodus of Gly through transport system L.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42495/1/10545_2005_Article_BF01800357.pd