Surrounding Incubation Medium Research Articles

Binding of Muscimol-Conjugated Quantum Dots to GABAC Receptors

Functionalization of highly fluorescent CdSe/ZnS core-shell nanocrystals (quantum dots, qdots) is an emerging technology for labeling cell surface proteins. We have synthesized a conjugate consisting of approximately 150-200 muscimols (a GABA receptor agonist) covalently joined to the qdot via a poly(ethylene glycol) (PEG) linker (approximately 78 ethylene glycol units) and investigated the binding of this muscimol-PEG-qdot conjugate to homomeric rho1 GABAC receptors expressed in Xenopus oocytes. GABAC receptors mediate inhibitory synaptic signaling at multiple locations in the central nervous system (CNS). Binding of the conjugate was analyzed quantitatively by determining the fluorescence intensity of the oocyte surface membrane in relation to that of the surrounding incubation medium. Upon 5- to 10-min incubation with muscimol-PEG-qdots (34 nM in qdot concentration), GABAC-expressing oocytes exhibited a fluorescent halo at the surface membrane that significantly exceeded the fluorescence of the incubation medium. This halo was absent following muscimol-PEG-qdot treatment of oocytes lacking GABAC receptors. Incubation of the oocyte with free muscimol (100 microM-5 mM), PEG-muscimol (500 microM), or GABA (100 microM - 5 mM) substantially reduced or eliminated the fluorescence halo produced by muscimol-PEG-qdots, and the removal of GABA or free muscimol led to a recovery of muscimol-PEG-qdot binding. Unconjugated qdots and PEG-qdots that lacked conjugated muscimol neither exhibited significant binding activity nor diminished the subsequent binding of muscimol-PEG-qdots. The results indicate that muscimol joined to qdots via a long-chain PEG linker exhibits specific binding activity at the ligand-binding pocket of expressed GABAC receptors, despite the presence of both the long PEG linker and the sterically bulky qdot.

Hydrolysis of hexose pentaacetate esters in rat pancreatic islets

The pentaacetate esters of selected hexoses were recently found to stimulate insulin release. The kinetics of their hydrolysis was now investigated in both rat pancreatic islet homogenates and intact islets. In islet homogenates, the hydrolysis of α- d-glucose pentaacetate, as judged from the measurement of acetate production, displayed a pH optimum of 7.4 and a K m for the ester of 0.95 mM. At pH 7.4, the reaction velocity was about 5 times higher than the rate of α- d-glucose pentaacetate hydrolysis by intact islets, as judged from the ester-induced increase in the acetate content of both the islet and surrounding incubation medium. Comparable results were obtained in intact islets exposed to either β- l-glucose pentaacetate or β- d-galactose pentaacetate. The ester content of the islets after 120 min incubation was close to 0.1 nmol/islet, yielding an apparent intracellular concentration at least one order of magnitude higher than the extracellular concentration (1.7 mM). These findings indicate that hexose esters that either stimulate insulin release or fail to do so are equally well taken up and hydrolyzed by islet cells. They are compatible, therefore, with the view that the insulinotropic action of some of these esters may be favored by the catabolism of their hexose moiety, although some other mechanisms for stimulation of insulin release must be operative in the case of β- l-glucose pentaacetate.

Factors affecting endo-β-mannanase activity in the endosperms of fenugreek and carob seeds

AbstractEndosperms of fenugreek and carob which have been leached following their isolation exhibit increased activity of endo-β-mannanase, part of which is released into the surrounding incubation medium. This activity was suppressed by the addition to the endosperms of abscisic acid or endosperm/seed coat leachate. The leachate from carob inhibited the increase of endo-β-mannanase activity in fenugreek and that from fenugreek inhibited enzyme activity in carob but more weakly; neither of these leachates inhibited the production of α-amylase in wheat endosperm. Polyethylene glycol-induced stress on non-leached endosperms inhibited endo-β-mannanase production, but not when the stress was applied following endosperm leaching. However, the stress did reduce the amount of enzyme released into the surrounding incubation medium. Several isoenzymes of mannanase could be detected on isoelectric focusing activity gels in the endosperms of both fenugreek and carob, but their pis differed between the two species. When activity was suppressed by ABA or leachate, all pl forms declined equally.

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.

Metabolism and DNA binding of 1-nitro[14C]pyrene by isolated rabbit tracheal epithelial cells

The metabolism of 1-nitro[14C]pyrene (1-NP) and the binding of its reactive intermediates to DNA and protein was examined in rabbit tracheal epithelial cells. The tracheal cells isolated by two protease digestion methods had normal cellular morphology and viability. The digestion method which demonstrated the highest rate of 1-NP metabolism and DNA binding used a 1 h incubation of the tracheas with 1% protease at 37 degrees C. Metabolites from the incubation medium and cell lysates were extracted, analyzed and quantitated by h.p.l.c. The majority of the metabolites produced by tracheal cells were EA:AC extractable and were released to the surrounding incubation medium. The predominant metabolites identified and quantified in the medium were the ring oxidation products, a 1-NP-diol, 1-NP phenols (6- or 8-OH-1-NP), 10-OH-1-NP and 3-OH-1-NP. Major metabolites formed by nitroreduction were NAAP and 1-AMP. The metabolites retained by tracheal cells were qualitatively identical to those identified in the medium but were present at one-seventh to one-tenth of the amounts. The rate of both 1-NP metabolism and DNA binding was very high. After 4 h, 62% of the 1-NP was metabolized and 198 DNA adducts/10(6) nucleotides were formed. The rate of [14C]1-NP-DNA adducts formation was highest at 30 min of incubation (124 adducts/10(6) nucleotides/h). These high rates of metabolism and DNA binding are consistent with the possibility that tracheal cells may be a particular target tissue for tumor induction by nitrosubstituted polycyclic aromatic hydrocarbons associated with diesel and other combustion emission particles. The preponderance of highly mutagenic 1-NP phenols as metabolites suggest that ring oxidation as well as nitroreduction may result in intermediates which form DNA adducts.

Intracellular Messengers in Stimulus-Secretion Coupling of Pancreatic Acinar Cells

Regulation of steady-state free Ca2+ concentration at rest and at stimulation have been studied in isolated permeabilized pancreatic acinar cells by measuring the free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. Ca2+ transport mechanisms have been further characterized in subcellular membrane fractions by measuring 45Ca2+ uptake into membrane vesicles and protein phosphorylation using polyacrylamide gel electrophoresis. (a) In permeabilized isolated acinar cells from exocrine glands, inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from endoplasmic reticulum. (b) Secretagogue-induced Ca2+ release from permeabilized cells is accompanied by increased production of IP3. At rest, steady-state free Ca2+ concentration is regulated at 4 X 10(-7) mol/L by the rough endoplasmic reticulum (RER). Ca2+ uptake into this pool is promoted by a (Ca2+ + Mg2+)-ATPase, and is dependent on cations and anions in the incubation medium in the order K+ greater than Na+ greater than Li+ greater than choline+ and Cl- greater than Br- greater than SO4(2-) = NO3- greater than I- greater than cyclamate- greater than SCN-, respectively. Similarly, Ca2+-stimulated 32P incorporation from [gamma 32P]ATP into a 130 kD protein intermediate of (Ca2+ + Mg2+)-ATPase, as well as 32P liberation, indicating (Ca2+ + Mg2+)-ATPase activity, are cation dependent. While 32P incorporation is highest in the presence of choline, 32P liberation is higher with K+, as compared with Na+ or choline, indicating that K+ ions facilitate dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cytosolic free Ca2+ concentration in acinar cells of rat pancreas.

Ca2+ uptake into isolated exocrine pancreatic cells with highly permeable plasma membrane was determined by measuring the decrease in free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. In the presence of Mg-ATP and respiratory substrates the free Ca2+ concentration of the incubation medium decreased rapidly after addition of leaky cells until a stable medium free Ca2+ concentration of 4.2 +/- 0.1 X 10(-7) mol/l was obtained. Changes in the medium free Ca2+ concentration at steady state by addition of Ca2+ or EGTA were buffered by cellular uptake or release, respectively, until the steady-state free Ca2+ concentration was reestablished. When nonmitochondrial Ca2+ uptake was determined in the presence of a combination of mitochondrial inhibitors (10(-5) mol/l antimycin, 5 X 10(-6) mol/l oligomycin, and 10(-2) mol/l azide), the rate of uptake was considerably reduced, while the steady-state concentration was unaltered. In contrast, mitochondrial uptake that could be observed in the presence of the ATPase inhibitor vanadate (2 X 10(-3) mol/l) proceeded at the same rate as the control, but the minimal medium free Ca2+ concentration reached was 2.4 +/- 0.1 X 10(-7) mol/l higher than the control. Addition of secretagogues at steady-state free Ca2+ concentration resulted in a Ca2+ release of 0.73 +/- 0.08 nmol/mg protein. The increase in medium free Ca2+ concentration was entirely transient and followed by reuptake to the prestimulation level. The data indicate that a cytosolic free Ca2+ concentration of 4 X 10(-7) mol/l can be regulated in pancreatic acinar cells by a nonmitochondrial Mg2+-dependent Ca2+ pool.

A role for ?-galactosidase in the degradation of the endosperm cell walls of lettuce seeds, cv. Grand Rapids

Isolated endosperms of Grand Rapids lettuce (Lactuca sativa L.) seeds undergo extensive cell-wall degradation and sugars are released into the surrounding incubation medium. One sugar so released is galactose. α-Galactosidase (EC 3.2.122) is present at the same level in both dry and imbibed isolated endosperms and is responsible for the release of galactose. However, this enzyme does not act upon the native endosperm cell wall, but requires first its partial hydrolysis and the production of oligomers by the action of endo-β-mannanase (EC 3.2.1.787). Galactose is then cleaved from these oligomers, allowing their further subsequent hydrolysis by endo-β-mannanase. Thus α-galactosidase and endo-β-mannanase act cooperatively to effect the hydrolysis of the lettuce endosperm cell walls.

The stimulus-secretion coupling of glucose-induced insulin release. LIII. Calcium-dependency of the cyclic AMP response to nutrient secretagogues.

Above a threshold value in excess of 5.6 mM, D-glucose increases the amount of cyclic AMP measured by radioimmunoassay in pancreatic rat islets and their surrounding incubation medium. As judged from the cyclic AMP content of islets exposed to isobutylmethylxanthine (1.0 mM), the glucose-induced increment in the rate of cyclic AMP generation represents a rapid and sustained phenomenon. The stimulant action of glucose on cyclic AMP accumulation is mimicked by L-leucine, and L-glutamine, these amino acids acting synergistically of one another. Trifluoperazine slightly decreases but fails to abolish the effect of glucose. In the absence of extracellular Ca2+, however, the cyclic AMP response to D-glucose, L-leucine and/or L-glutamine is severely impaired. These findings are compatible with the view that an increase in the generation rate of cyclic AMP participates in the process of nutrient-stimulated insulin release. This increase could be secondary to the nutrient-induced accumulation of Ca2+ in the islet cells leading to activation of adenylate cyclase by calmodulin.

The in vitro incorporation of [U- 14C] valine into fat body protein of the larva of the blowfly, Calliphora erythrocephala

The incorporation of [U- 14C] l-valine into fat body isolated from the larva of the blowfly, Calliphora erythrocephala, has been studied in vitro. The effect of various concentrations of l-valine and of a mixture of amino acids was studied at pH 6·5. The highest incorporation rates were obtained with fat body isolated from 4-day-old larvae, the rate falling off rapidly as the larvae became older. It was found that the fat body synthesized protein which was then released into the surrounding incubation medium. During the course of incubation the specific activity of this released protein increased rapidly until its level was many times that of the structural protein of the fat body.