Dale L. Oxender

The regulation of neutral amino acid transport in mammalian cells

II1. Regu la t ion of t r anspor t ac t iv i ty by amino acid avai lab i l i ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 A, Regula t ion of System A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 B. Regula t ion of System L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 1. Kinet ic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 2. Gene t i c approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 C, Regula t ion of System N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

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

SUMMARY I. 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 ° and for glucose or zero sugar at o °. 2. From these data, using a simplified form of the theoretical analysis of RECEN AND MORGAN 18, it is shown that the rate of movement across the membrane of the substrate-carrie r complex is greater than that of the free carrier, that is, the movement of carrier is facilitated by the substrate. 3- A lower limit of 2.8 is derived for the ratio of these rates of movement. 4. Evidence is presented that the aldoses and ketoses share a common carrier.

Cellular Localization of Leucine-Binding Protein from Escherichia coli

Rabbit antibody against lecine-binding protein isolated from Escherichia coli K-12 has been prepared. This antibody has been used in conjunction with enzyme-labeled antibody to allow an immunocytochemical localizati of leucine-binding protein in the Escherichil coli cell. This protein appears to be present only in the envelope and not in the cytoplasm.

Complement-mediated Cytotoxic Effects on Pancreatic Islets With Sera from Diabetic Patients

Sera from 11/30 patients with juvenile-onset-type diabetes (JOD) had cytotoxic effects on isolated pancreatic islets from hamsters when guinea pig complement was present. The cytotoxic activity could be readily eliminated by heating the guinea pig complement at 56°C for 30 min, thus demonstrating a complement-dependent process. Complement-dependent cy totoxicity toward pancreatic islets was not observed with sera from 11 patients with maturity-onset-type diabetes, 4 nondiabetic hospital patient controls, and 28 healthy adult subjects. The presence of circulating complement-mediated cytotoxic factor(s) toward isolated islets indicates the potential importance of autoimmune pathogenic damage to pancreatic islets in some patients with diabetes.

Mitogen-activated Protein Kinase Regulates Transcription of the ApoCIII Gene INVOLVEMENT OF THE ORPHAN NUCLEAR RECEPTOR HNF4

The transcriptional regulation of the apoCIII gene by hormonal and metabolic signals plays a significant role in determining plasma triglyceride levels. In the current work we demonstrate that the apoCIII gene is regulated by the mitogen-activated protein (MAP) kinase signaling pathway. In HepG2 cells, repression of MAP kinase activity by treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused a 5–8-fold increase in apoCIII transcriptional activity. Activation of MAP kinase by phorbol ester treatment caused a 3–5-fold reduction in apoCIII transcription. The region of the apoCIII promoter responsible for this regulation was mapped in transiently transfected HepG2 cells to a 6-base pair element located at −740. The major protein binding to this site was identified as the nuclear hormone receptor HNF4. An increase in HNF4 mRNA and protein levels was observed in HepG2 cells after treatment with PD98059, indicating that the MAP kinase pathway regulates the expression of the HNF4 gene. These findings demonstrate that the apoCIII gene can be regulated by signals acting through the MAP kinase pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF4.

A novel phosphate-regulated expression vector in Escherichia coli

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.

The regulation of neutral amino acid transport by amino acid availability in animal cells

Animal cells regulate the activities of neutral amino acid transport Systems A and L to keep the intracellular supply of amino acids relatively constant. Transport System A activity increases dramatically in response to starvation of all the amino acids. Transport System L activity increases in response to starvation of a single substrate such as leucine. The mechanism of regulation appears to be different for Systems A and L.

BINDING PROTEINS AND MEMBRANE TRANSPORTfn1

The recent studies have clearly established two types of active transport systems. One type is membrane-bound and can be observed in membrane vesicles and the other type is osmotic-shock-sensitive and requires binding proteins to produce active transport. It appears that the membrane-bound systems derive cellular energy from the energy-rich membrane state which can be formed from respiration or ATP-hydrolysis, while the binding protein systems are more directly coupled to phosphate bond energy derived from glycolysis or oxidative phosphorylation. The following conclusions concerning the role of the binding proteins are offered: 1. The binding proteins are present in relatively large amounts (approximately 10(-6) or 10%-5) M) and appear to reside in the periplasmic space. 2. They do not appear to be involved in solute translocation steps, although they cantain a second binding site that could interact with membrane components. 3. The binding proteins appear to increase the affinity of the transport system for the solute by interacting with a membrane component. This may substrate for the membrane transport system.

Isolation and Characterization of Membrane Binding Proteins

The term binding protein in the field of membrane transport refers to the group of relatively low molecular weight proteins possessing a reversible binding activity for solutes of specific transport systems. Most of these proteins have been isolated from gram-negative bacteria by a cold osmotic shock treatment. No enzymatic function has been demonstrated for these proteins. The mild shock treatment of gram-negative bacteria also removes the periplasmic enzymes (Heppel, 1971), so called because they appear to be located in the “periplasm,” i.e., the space between the cytoplasmic membrane and the cell wall (Mitchell, 1961). A large body of data has accumulated to suggest that the binding proteins act as the recognition site for active transport systems. Recent genetic evidence indicates that these proteins play a direct role in solute transport.

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

SummaryThe 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.