R. Brian Beechey

Phosphate transport in intestinal brush-border membrane

In the small intestine of the rabbit the process of Na+-dependent uptake of phosphate occurs only at the brush-border of duodenal enterocytes. Li+ can replace Na+. The process is activated when either K+, Cs+, Rb+, or choline is present in the intravesicular space. The presence of membrane-permeable anions is essential for maximum rates of phosphate transport. We conclude that the mechanism of the phosphate carrier is electrogenic at pH 6–8, probably two Na+ moving with each H2PO4−. This. will lead to the development of a positive charge within the vesicle. The variation of theKm for H2PO4− with pH is thought to be the consequence of the affinity of the carrier protein for H2PO4− increasing as the pH increases. Polyclonal antibodies against membrane vesicles isolated from rabbit duodenum, jejunum, and ileum were prepared. The antibodies raised against the ileum and jejunum both activated the phosphate transport process, while the anti-duodenum antibody preparation inhibited phosphate transport.

Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs

1. Brush border membrane vesicles were prepared from lamb enterocytes. These were used to study the changes in the enzyme contents and the transport capacities which occur during the change from a milk to a roughage diet. 2. Na+-dependent transport of D-glucose was present in all regions of the small intestine of pre-ruminant lambs and absent in ruminants. 3. Na+-dependent transport of L-proline was present in all regions of the small intestine irrespective of the age of the animal. 4. Phosphate transport was seen only in the presence of a transmembrane pH gradient (acid outside). The transport was not stimulated by either Na+ or K+. The transport capacity increases 2-fold as the animal becomes ruminant. 5. The activities of lactase and maltase diminished with age. Alkaline phosphatase and aminopeptidase N activities remain constant. Sucrase activity cannot be detected in lambs of any age.

The extraction and reconstitution of the α-cyanocinnamate-sensitive pyruvate transporter from castor bean mitochondria

The pyruvate carrier from castor bean mitochondria has been solubilized with Triton X-114 and partially purified using hydroxyapatite column chromatography. SDS-polyacrylamide gel electrophoresis of the hydroxyapatite-eluate showed that there were 6 major protein bands of Mr, 74kDa, 66kDa, 34kDa, 32kDa, 30kDa 12kDa. When the eluate was reconstituted into liposomes it was shown to catalyze a pyruvate exchange reaction which was sensitive to N-ethyl maleimide and a series of analogues of alpha-cyanocinnamate. The characteristics of this pyruvate exchange activity are similar to that found in intact mitochondria, and it is concluded that one or more proteins in the hydroxyapatite-eluate correspond to the pyruvate carrier.

Identification of the phosphate-translocator from maize mesophyll chloroplasts

Intact maize mesophyll chloroplasts have been isolated in yields of up to 10 mg of chlorophyll per preparation. The chloroplasts were able to reduce 3-phosphoglycerate at a rate of 2.4 mumol of oxygen/min/mg of chlorophyll. This activity was inhibited by preincubating the intact chloroplasts with pyridoxal 5-phosphate. Chloroplast envelopes have been prepared and the protein profile has been obtained on SDS-polyacrylamide gels. The phosphate-translocator from the chloroplast envelope has been identified as a 30kDa polypeptide.

THE USE OF AMPHIPATHIC MALEIMIDES TO STUDY MEMBRANE-ASSOCIATED PROTEINS

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD-β-hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.

Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane

The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S is freely permeable to both TPP+ and hydroxymethylinulin. Tuv and Cuv are able to exclude these compounds. EDTA treatment was necessary prior to measuring membrane potential in Tuv and Cuv. Under conditions where delta phi could be measured, uncouplers acted to dissipate delta phi with equal potency in all strains. Uncoupler resistant proline uptake in Tuv and Cuv was abolished by EDTA treatment. Transduction experiments with phage P1 showed that uncoupler resistance could be transferred from Tuv to Doc S. Such transductants were no longer sensitive to novabiocin. The gene for uncoupler resistance cotransduced with the gene pyrE (82 min). Plating efficiency experiments with P1 suggests that detergent sensitivity in Doc S arises from an rfa (81 min) mutation. This mutation is no longer present in Tuv.

Castor Bean Mitochondrial Protein Synthesis in Response to Temperature-Induced Stress

Higher plants when exposed to elevated temperatures respond by rapid and increased expression of sets of proteins known as heat-shock proteins1. These proteins are thought to primarily protect the cellular systems from lethal thermally-exerted damage. Mitochondria have been implicated as targets of the heat-shock response on the basis of the observations that agents such as uncouplers of oxidative phosphorylation and inhibitors of respiratory chain cause induction of the heat-shock proteins2. The findings that isolated corn (Zea mays) mitochondria when exposed to elevated temperatures express in vitro novel polypeptides3, 4, also suggests that the organelles have a role to play in heat-shock response. Here we present some preliminary findings of the studies on the direct effects of temperature shifts on the protein synthesis by isolated, functional castor bean (Ricinus communis) mitochondria.