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Cysteine string protein (Csp) is a secretory vesicle protein previously demonstrated to be required for Ca2+-regulated exocytosis in neurons and endocrine cells. It has been suggested to function by regulating voltage-gated Ca2+ channels or, alternatively, to have a more direct effect on the regulated exocytotic machinery. Here we demonstrate the expression of Csp in mammary epithelial cells and in the KIM-2 mammary cell line. In KIM-2 cells, Csp was found to be associated with a population of small vesicles and showed partial co-distribution with the vesicle protein cellubrevin. KIM-2 cells do not express detectable levels of voltage-gated Ca2+ channels, ruling these out as a site of action. Using the release of transfected growth hormone (GH) as an assay of secretion, we found that GH is secreted in an exclusively constitutive manner from KIM-2 cells. Overexpression of Csp1 inhibits regulated exocytosis in other cell types but has no effect on constitutive GH release by KIM-2 cells. These results suggest that Csp does not have a major function in constitutive exocytosis.

The transport of acetate and formate into plasma membrane vesicles derived from the basolateral face of the ovine parotid acinar cell has an absolute requirement for an anion to be present within the intravesicular space: bicarbonate, formate, acetate, propionate, and butyrate support the uptake of either acetate or formate. A pH gradient across the vesicle membrane, pHi 7.4, pH0 5.5, enhances the uptake of formate, but not acetate. There is no direct relationship between the rate of exchange and the degree of protonation of formate or acetate in the extravesicular medium. The process is saturable and can be inhibited by a range of functional group reagents. When mannitol is the main external osmoticum, the uptake of acetate and formate is still rapid; thus, no other ions are involved in the process apart from the external formate or acetate and the intravesicular anion. This activity could play a major role in the provision of energy in ruminant tissues., (Copyright 1999 Academic Press.)

Inorganic orthophosphate (Pi) transport, using 32P-labelled orthophosphate as tracer, by lactating rat mammary tissue has been examined using both tissue explants and the intact perfused gland. Pi uptake was predominantly via a Na+ -dependent pathway. Li+, however, unlike choline, was able to partially substitute for Na+. In addition, Pi release from tissue explants preloaded with 32Pi was stimulated by reversing the Na+ gradient. Thus transferring mammary explants from a buffer containing Na+ to one which was Na+ free (choline replacement) doubled the Pi efflux rate constant. The uptake of Pi by tissue explants was saturable with respect to external Pi, having apparent K(m) and V(max) values of 1.13 mM and 3.36 mmol (kg cell water)-1 (15 min)-1, respectively. The stimulation of Pi uptake by tissue explants by external Na+ was also saturable; the K(m) for Na+ was 9.7 mM. These results, taken together, suggest that the Na+ -dependent pathway is a Na+ -Pi cotransport mechanism. The transport of Pi by the perfused lactating rat mammary gland was examined using a rapid, paired-tracer dilution technique. Pi uptake by the perfused gland was found to be Na+ dependent and displayed saturable kinetics. The results suggest that the Na+ -Pi cotransporter is situated at the basolateral aspect of the secretory cells. The release of Pi from preloaded tissue explants was trans-accelerated by external Pi but not by Cl- or SO4(2-). However, external Pi stimulated Pi efflux with low affinity.

The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence.

There is a rapid stimulation of the rate of efflux of 3-O-methyl D-glucose from intact lactating rat mammary tissue slices in response to a change in the Na(+)-gradient across the basolateral plasma membrane. RNA extracted from lactating mammary glands contains a 4 kb transcript which hybridises with the cDNA for rabbit intestinal SGLT1. A fraction of the mammary gland cells which is enriched in endoplasmic, Golgi and plasma membranes contains a protein that is immunologically similar to the Na(+)-dependent D-glucose symporter present in the plasma membrane of polarised epithelial cells, e.g., rabbit enterocytes, ovine enterocytes and parotid acinar cells.

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

We have shown previously that the activity and abundance of the intestinal Na+/glucose cotransporter (SGLT1) declines dramatically during the postnatal development of lambs, and that it can be restored in the intestine of ruminant sheep by intra-luminal infusion of D-glucose. The work presented in this paper has followed the expression of the SGLT1 gene along the vertical and horizontal axes of the ovine small intestine during early development, using quantitative in situ hybridisation histochemistry. Along the vertical axis, SGLT1 mRNA was first detectable just below the crypt-villus junction and rose rapidly to a peak level approx. 150 microns above this point. After reaching a maximum, the amount of message gradually declined towards the villus tip. This pattern of mRNA accumulation along the crypt-villus axis was similar in all intestinal positions and age groups. Along the length of the small intestine (horizontal axis), a decline in the level of SGLT1 mRNA was observed first in the distal intestine. This decrease in SGLT1 mRNA was significant in the intestine (75% of length) of 5-week-old lambs when compared to tissue taken from 25 and 50% of length (P < 0.01 and P < 0.02, respectively). However, the observed fall in the expression of this gene during weaning did not coincide with the fall in activity and amount of SGLT1. In adult animals, where the activity of SGLT1 is very low, the amount of message was greatly reduced. This work supports the finding that the expression of SGLT1 is primarily controlled at the post-transcriptional level during the postnatal development of ovine intestine.

The Na+/bile salt co-transporter of the pig ileal brush border membrane has been expressed in Xenopus laevis oocytes. Injection of pig ileal poly (A)+ RNA into oocytes resulted in the functional expression of an Na(+)-gradient-stimulated taurocholate uptake within 2-5 days. The expressed Na(+)-dependent taurocholate uptake exhibited saturation kinetics (apparent Km of 48 microM), and displayed similar competitive substrate inhibition by taurodeoxycholate as the native brush border Na+/bile salt co-transporter studied in pig ileal brush border membrane vesicles. Interestingly, injection of pig proximal and mid intestinal poly (A)+ RNA into oocytes also resulted in the expression of the Na+/bile salt co-transporter, though the Na(+)-dependent transport of bile salts does not occur in brush border membrane vesicles (BBMV) isolated from pig proximal and mid intestine. This suggests that the mRNA coding for the co-transporter is present in the enterocytes lining the whole length of the small intestine, but that the function is only expressed in the brush border of the distal small intestine. The transport of D-glucose into BBMV, and the transport of methyl-alpha-D-glucopyranoside (a non-metabolizable hexose derivative) into oocytes were used throughout the study as methods of confirming the integrity of vesicles and oocytes.

A procedure is described for the preparation of basolateral membrane vesicles from the acinar cells of the sheep parotid gland. The ouabain-sensitive K(+)-activated phosphatase activity was enriched 30-fold over the tissue homogenate; 45% of this activity was recovered in the final membrane fraction. The presence of membranes from other organelles was negligible. Evidence is presented for the location of Na(+)-dependent symporters for phosphate and D-glucose on the basolateral membrane.

The transport of phosphate in intestinal brush-border membrane and parotid basolateral membrane vesicles isolated from sheep maintained on high and low phosphate diets have been studied. The mechanism of the transport of phosphate in the intestine is via a proton symporter whilst in the parotid gland it is effected by a Na+ coupled transporter. In sheep fed a low-P diet there is no change in the capacity of the parotid basolateral membrane to transport phosphate into the parotid end piece cells. This is in marked contrast to the response of the enterocyte brush-border membrane, where there is a significant enhancement of the capacity of the membrane to transport phosphate. We conclude that in sheep the gut appears to play a major role in response to phosphate deprivation, by increasing the capacity to transport phosphate. This enhancement is not achieved by increases in the levels of circulating 1,25-dihydroxycholecalciferol.

A series of amphiphilic polymethlyenecarboxymaleimides 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 sitting 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 of D-beta-hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in the D-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell 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.

The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.

This study describes a simple and rapid method for the preparation of brush-border membrane vesicles from intestinal biopsies. The specific activities of sucrase, amino peptidase N, and alkaline phosphatase in these vesicles were the same as those in vesicles prepared from intestinal segments. The vesicles from all the regions of the small intestine can transport D-glucose in an Na+-dependent manner. The rates of transport of D-glucose presented here are far higher than previously reported. The method should have a wide applicability to studies of transport mechanisms and the distribution of transport processes within the intestine.

The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors. Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli. None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit. The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript. Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes. The 5' end of the wheat small-subunit precursor was linked with a consensus E. coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus. The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein. The bacterially synthesized small subunit precursor was purified to homogeneity. The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.

Dibutylchloromethyl tin chloride (DBCT) inhibits coupled and uncoupled respiration of mitochondria from potato tubers, cauliflower florets and etiolated mung bean hypocotyls with succinate and L-malate but not with external NADH or TMPD/ascorbate as substrates. Using potato and cauliflower mitochondria. DBCT at 200 pmole/mg of protein gives complete inhibition only in KCl-based media and at pH 6.8. DBCT has no effect on the internal pH of mung bean mitochondria, but does cause a decrease in the membrane potential. Electron transport through the alternative oxidase is not inhibited, neither is the ATP-synthase system. DBCT appears to interact with the functionally-distinct pool of ubiquinone associated with the oxidation of succinate and L-malate.

Bioenergetic properties of a mutant strain of Escherichia coli K12 designated TUV, which is resistant to the protonophoric uncoupling agent 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidiazole (TTFB) have been compared with those of its non-resistant parent, E. coli K12 Doc-S. Strain TUV grew and respired some 20-30% faster than strain Doc-S, and was cross-resistant to carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and triphenyltin, but not to 2,4-dinitrophenol. Phosphorus nuclear magnetic resonance demonstrated the TTFB-mediated collapse of the transmembrane pH gradient at identical rates in starved cells of both strains, indicating that uncoupler access and function were unimpaired in the mutant under these conditions. Strain TUV displayed enhanced uncoupler resistance and maintained intracellular pH and ATP levels only when respiring. On the other hand, strain TUV also showed increased resistance to novobiocin, implying that its outer wall permeability had been lowered. We suggest that the active resistance of strain TUV results from the exclusion of uncoupler by the interaction of inner and outer membrane components in a manner modulated by the degree of cellular energization.

Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the alpha and beta subunits and is equally distributed between them. The incorporation of o-ATP into the ATPase is reduced, and the irreversible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number, and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenosine-containing compound and the tripolyphosphate anion in a beta-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the beta-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.

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 the Km 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.

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme II (EIIGlc) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl alpha-D-glucopyranoside (alpha-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGlc. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate alpha-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent N-ethylmaleimide was used. Since only one activity-linked dithiol can be detected with sealed inverted membrane vesicles or intact cells and it is accessible to membrane-impermeable sulfhydryl reagents from both sides of the cytoplasmic membrane, we suggest that it is located in a channel constructured by the carrier and that the channel spans the membrane. A second dithiol, not essential for activity, is located near the outer surface of the cytoplasmic membrane.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

Cyanide added to isolated castor bean (Ricinus communis L.) mitochondria supplemented with ATP and succinate (or NADH) significantly enhanced the rate and extent of organellar protein synthesis. Cyanide stimulated mitochondrial protein synthesis in a dose-dependent manner with an optimum stimulation of over twofold at 1 millimolar cyanide. At this concentration of cyanide, the mitochondrial respiratory activity, in the presence of succinate (or NADH) and ADP was inhibited by 90%. The stimulatory effect of cyanide on mitochondrial translation was reflected in the increased synthesis of all the proteins synthesized within the organelle. Preliminary evidence indicates a role for the alternative, salicylhydroxamic acid-sensitive, oxidase in the cyanide stimulation of protein synthesis.

A simple device is described for efficient and reproducible electro-elution of DNA resolved in agarose gels. DNA (greater than 1 micrograms) is recovered with consistent yields of over 70% into preset elution volumes of 100 to 500 microliters.

A rapid sampling technique was used to follow nucleoside uptake by Trichomonas vaginalis. The results indicated that nucleoside uptake is biphasic with time. Adenosine, guanosine, and uridine uptake is carrier mediated, transported substrate is rapidly metabolised to nucleotides. Two separate carriers appear to exist, one which transports all nucleosides and a second which transports adenosine, guanosine and uridine. Both carriers have more than one binding site for nucleosides. The first carrier has sites for adenosine and pyrimidine nucleosides, and a separate site for purine nucleosides. The second carrier has a site for adenosine and uridine and a separate site for guanosine. Adenosine uptake could not be completely inhibited by nitrobenzylthionucleosides. The rate of nucleoside uptake by T. vaginalis is sufficient to sustain growth.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.