T. Hastings Wilson

Development of structure and function in the mammalian yolk sac. I. Developmental morphology and vitamin B12 uptake of the rat yolk sac.

Abstract Morphological and physiological aspects of differentiation have been analyzed in the visceral splanchnopleure of the inverted yolk sac placenta of the albino rat between 13 days and term. The visceral yolk sac is known to be involved in the transfer of antibodies from the maternal to the fetal blood. The absorptive cells of the visceral endoderm possess ultrastructural features characteristic of protein-absorbing cells. The appearance of the free surface of these simple columnar cells suggests motility. It is primarily modified to form long, branching microvilli that often seem to be fusing. Invaginations of the free surface originate between microvilli, and most probably connect with an extensive canalicular system of smooth membranes located in the superficial cytoplasm. The inner surface of both the invaginations and the canaliculi possesses a filamentous coat that may represent a vast internal surface for the attachment of proteins. Morphological and physiological evidence suggests that large absorptive vacuoles arise as dilatations of the canaliculi. The content of the more deeply located vacuoles is more condensed, and they may be called absorptive droplets. Thus, a superficial system of smooth membranes is directly involved in the uptake of macromolecules, such as antibodies and other proteins. During this period of gestation various morphological changes occur that may affect absorptive activity considerably. As gestation proceeds, the microvilli become shorter and less branched; the smooth membrane system is altered; and intercellular associations change as large dilatations appear in the intercellular space of the absorptive epithelium. In addition, there is a thickening of the placental barrier that intervenes between the yolk sac (or uterine cavity) and the vitelline circulation. The present findings indicate that the visceral yolk sac possesses a highly specific mechanism for accumulating considerable vitamin B12 and B12-intrinsic factor complex in vitro. The parietal wall lacks this mechanism. The capacity to concentrate vitamin B12 and B12-intrinsic factor both in vivo and in vitro is greater at 13 days than near term when determined on the basis of radioactivity per unit weight. This suggests a decrease in absorptive capacity per cell as gestation proceeds. The reduced absorptive activity of each cell is, however, more than counterbalanced by a seventyfold increase in the mass of this placental membrane. Microscopic examination revealed that the morphology of the visceral yolk sac is well preserved during a 60-minute period of in vitro incubation. Experimental findings in vitro indicate that although the visceral splanchnopleure can accumulate a large macromolecular complex, such as vitamin B12-IF, it lacks the capacity to concentrate small molecules, such as l -valine and α-methylglucoside, against a concentration gradient. This evidence suggests that this placental membrane in the rat may be highly specialized for the transfer of macromolecules.

Cation-sugar cotransport in the melibiose transport system of Escherichia coli.

The entry of Na+ or H+ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 microM Na+. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na+ concentration in the incubation medium. No TMG-stimulated Na+ movement was observed in uninduced cells. In an alpha-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na+ uptake was observed in melibiose-induced cells on the addition of melibiose, thiodigalactoside, methyl-alpha-galactoside, methyl-beta-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na+ when this cation is present in the incubation medium. Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na+ or Li+ the addition of melibiose or methyl-alpha-galactoside resulted in marked alkalinization of the external medium (consistent with H+-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-beta-galactoside gave no proton movement under these conditions. When Na+ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na+ with TMG or melibiose carries into the cell a positive charge (Na+) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.

Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli

A study has been made of the sugar substrate specificities and the cation specificities of the melibiose transport system of Escherichia coli. The following beta-galactosides were found to be transported: lactose, L-arabinose-beta-D-galactoside, D-fructose-beta-D-galactoside, o- and p-nitrophenyl-beta-D-galactosides. These beta-galactosides were cotransported with Na+ but not with H+. The alpha-galactosides raffinose, melibiose and p-nitrophenyl-alpha-galactoside were transported with either H+ or Na+. Of the monosaccharides tested D-galactose could use either Na+ or H+ for cotransport whereas D-fucose, L-arabinose and D-galactosamine could use only Na+. The sugar specificity requirements for H+ cotransport are therefore more exacting than those for Na+ cotransport.

Co-transport of Na+ and methyl-β-D-thiogalactopyranoside mediated by the melibiose transport system of Escherichia coli

Na+-dependent transport of methyl-β-D-thiogalactopyranoside (TMG) mediated by the melibiose transport system was investigated in Escherichia coli mutants lacking the lactose transport system. When an inwardly-directed electro-chemical potential difference of Na+ was imposed across the membrane of energy depleted cells, transient uptake of TMG was observed. Addition of TMG to cell suspensions under anaerobic conditions caused a transient acidification of the medium. This acidification was observed only in the presence of Na+ or Li+. These results support the idea that TMG is taken up by a mechanism of Na+-TMG co-transport via the melibiose transport system in Escherichia coli.

Counterflow of galactosides in Escherichia coli.

Abstract Counterflow of β-galactosides in metabolically poisoned cells of Escherichia coli was systematically examined. In one type of experiment (“entrance counterflow”), azide-treated cells were preloaded with a galactoside, cells separated from the medium and finally resuspended in thio[ 14 C]methyl-β- d -galactopyranoside ([ 14 C]TMG). The concentration of radioactive sugar within the cell rose to levels well above that in the medium (due to the inhibition of efflux) and subsequently fell to the concentration of the external medium (as the preloaded sugar was lost from the cell). Preloading the cell to different levels of non-radioactive sugar resulted in different heights of the counterflow curve, but the time to attain the peak of each was the same. Reduction in the number of membrane carriers resulted in a proportionate reduction in the initial rate of rise of the counterflow curve and also a time delay in the peak of the counterflow curve. The effect of the following factors on counterflow was investigated: various concentrations of [ 14 C]TMG for uptake, various substrates, temperature and p -chloromercuribenzoate. In a second type of experiment (“exit counterflow”), poisoned cells were first equilibrated with [ 14 C]TMG and then a high concentration of galactoside added to the external medium. Inhibition of influx without a corresponding effect on efflux results in a net loss of [ 14 C]TMG from the cell. The effects of different substrates and different levels of carrier were investigated.

ATP synthesis driven by a protonmotive force inStreptococcus lactis

SummaryAn electrochemical potential difference for hydrogen ions (a protonmotive force) was artificially imposed across the membrane of the anaerobic bacteriumStreptococcus lactis. When cells were exposed to the ionophore, valinomycin, the electrical gradient was established by a potassium diffusion potential. A chemical gradient of protons was established by manipulating the transmembrane pH gradient. When the protonmotive force attained a value of 215 mV or greater, net ATP synthesis was catalyzed by the membrane-bound Ca++, Mg++-stimulated ATPase. This was true whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Under these conditions, ATP synthesis could be blocked by the ATPase inhibitor, dicyclohexylcarbodiimide, or by ionophores which rendered the membrane specifically permeable to protons. These observations provide strong evidence in support of the chemiosmotic hypothesis, which states that the membrane-bound ATPase couples the inward movement of protons to the synthesis of ATP.

Inhibition of β-galactoside transport by substrates of the glucose transport system in Escherichia coli

Abstract 1. 1. The uptake by Escherichia coli (ML 308) of thiomethyl-β-galactoside and o-nitrophenyl-β-galactoside was inhibited by glucose, α-methylglucoside and other compounds which are substrates for the glucose transport system. This inhibition of β-galactoside transport was observed only in glucose-grown cells. Such cells possessed a five-fold greater α-methylglucoside transport capacity (max) than cells grown on other carbon sources, the affinity for uptake (t being the same in both types of cell. Growth of cells on carbon sources (other than glucose) which resulted in levels of catabolite repression and glucokinase equivalent to those in glucose-grown cells did not render the cells susceptible to inhibition. Although cells grown on glucose accumulated more α-methylglucoside and α-methylglucoside phosphate than cells grown on other carbon sources at a given extracellular concentration of α-methylglucoside these intracellular pools could be made equally large in casein amino acid-grown cells by increasing the extracellular concentration of α-methylglucoside. glucoside. Thus, a high intracellular level of α-methylglucoside and its phosphorylated derivative is not a sufficient condition for the production of cells sensitive to α-methylglucoside. 2. 2. β-Galactosides did not inhibit the uptake of α-methylglucoside, and α-methyl-glucoside was unable to cause “counterflow” of thiomethyl-β-galactoside. Such experiments indicate the lack of reciprocity between galactoside and glucose transport systems and put severe restrictions on the “common carrier” or transporter hypothesis. The entrance of thiomethyl-β-galactoside and o-nitrophenyl-β-galactoside into metabolically poisoned cells was inhibited by α-methylglucoside indicating that energy coupling is not involved in the inhibitory phenomenon. 3. 3. It is suggested that the product of the “y” gene is the membrane carrier per se and that substrate-carrier complex requires a factor which is common to many carbohydrate transport systems and limited in amount. Thus, α-methyl-glucoside-carrier complex when present in large amounts combines with a significant fraction of the common factor making it unavailable for the β-galactoside transport system.

Galactoside transport dissociated from proton movement in mutants of Escherichia coli

Abstract Two mutants of Escherichia coli have been described in which the transport of β-galactosides is partly uncoupled from the metabolic reactions which drive active transport. It is shown that the effective inflow of H + , caused by the addition of β-galactoside, is much less in these mutants than in the parental strains, and it is concluded that β-galactoside transport is partly uncoupled from H + transport.

The Evolution of Ion Pumps

The earliest problem faced by living cells was an unavoidable trend to swelling and lysis, as extracellular salts and water leaked through a semipermeable plasma membrane enclosing impermeant macromolecules. Two solutions countered this ever-present threat-a rigid cell wall to resist expansion and ion pumps to offset the passive influx with an active efflux. On the premise that ion pumps were the first solution, we have constructed an evolutionary sequence to account for the diversity of ion pumps found today.

Effect of lithium ion on melibiose transport inEscherichia coli

SummaryBoth Li+ and Na+ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li+ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li+ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by α-galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li+ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of α-galactosidase. Li+ was found to inhibit the entry ofp-nitrophenyl-α-d-galactoside, but notp-nitrophenyl-β-d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.