Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Alliánce Media Release - June 6, 1995

A media release from Inniskillin Wines announcing the launch of "Alliánce", a joint winemaking venture between Inniskillin and Jaffelin Wines of France.

Draft News Release - Great Lakes Water Quality Agreement

A News Release draft to be sent to "100 newspapers, radio and television stations (virtually all those with offices within 20 miles of the Lakes), make them available to the Press Gallery, special interest groups, trade publication and Mayors etc. of Great Lake-side communities". The release discusses the need for an upgrade to "the 1972 Canada-U.S. Great Lakes Water Quality agreement". Within the document, O'Sullivan is quoted that the agreement "should be upgraded to become a treaty with the United States, so that after all the effort which has already been put into tyring to clean up the Great Lakes we the provision which provides for cancellation by either party giving twelve months (notice) to do so". The total report is 61 pages in length.

Press Release - Youth for Diefenbaker, 1967

A press release from Youth for Diefenbaker, 1 August 1967. The release reads: "Hamilton Student Appointed Youth for Dief Representative. The youngest delegate to the conservative leadership convention, 15 year old Sean O'Sullivan of Hamilton, has been named Western Ontario representative of the Youth for Diefenbaker movement. This movement was formed in Ottawa to engage support of Canada's young conservatives for John Diefenbaker at the leadership convention. According to Mr. O'Sullivan, there is growing support for Mr. Diefenbaker and he will have an announcement to make in that regard at a press conference on Friday August Fourth at the Connaught Hotel."

Press Release - Great Lakes and St. Lawrence Seaway Assignment, 25 October 1973

A press release assigning Sean O'Sullivan to study the future of the Great Lakes and St. Lawrence Seaway.

Indenture of Release to Woodstock and Lake Erie Railway from Executors of Samuel Zimmerman

Indenture of release regarding a loan of 12, 500 pounds which Samuel Zimmerman (deceased) is said to have lent and advanced to the Woodstock and Lake Erie Railway and Harbour Company. This is put forward by the executors who include: Joseph A. Woodruff, Richard Woodruff, John L. Ranney and Richard Miller. This document releases and exonerates the Railway Company from any debts to the executors. [The outside of the document says 1857, but the actual date is Feb. 10, 1858].

Indenture of Release of All Existing Contracts and Agreements with Woodstock and Lake Erie Railway Co.

Indenture of release stating that all existing contracts and agreements made by the Woodstock and Lake Erie Railway Co. are cancelled. This release was between the executors of Samuel Zimmerman's will and the Woodstock and Lake Erie Railway Co. February 10, 1858.

Indenture of Release between Richard Miller and others

Indenture of release by executors between Richard Miller, John L. Ranney and Richard Woodruff, all of St. Catharines and Joseph A. Woodruff of Clifton, all executors of the will of Samuel Zimmerman of the first part and The Great Western Railway Company of the second part. Parties of the second part have exonerated parties of the first part to undertake and complete the Sarnia branch of the railway. Also, parties of the first part believe they are entitled to compensation as the death of Zimmerman was caused while travelling on one of the carriages belonging to said company. A sum of $150,000 was agreed upon to be paid to the executors by the Great Western Railway Company, September 7, 1858.