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Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

Legerton, Timothy L. and Kanamori, Keiko and Weiss, Richard L. and Roberts, John D. (1983) Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy. Biochemistry, 22 (4). pp. 899-903. ISSN 0006-2960. doi:10.1021/bi00273a029.

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The nitrogen-15 chemical shift of the N1 (7)-nitrogen of ^(15)N-labeled histidine and the half-height line widths of proton-coupled resonances of the δ- and ω,ω-nitrogens of ^(15)N-labeled arginine and of the a-nitrogens of ^(15)N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain estimates of intracellular pH. For intracellular ^(15)N-labeled histidine, the N1 (T)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the δ- and ω,ω-nitrogens of [^(15)N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 ± 0.4 while the cytoplasmic pH is 7.15 ± 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.

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Additional Information:© 1983 American Chemical Society. Received July 30, 1982. This work was supported by Grant PCM78-19030 from the National Science Foundation (to R.L.W.), by a Biomedical Research Support grant to the University of California, Los Angeles, by US. Public Health Service Research Grant GM-11072 from the Division of General Medical Sciences, and by the National Science Foundation (K.K. and J.D.R.). T.L.L. was supported by US. Public Health Service National Research Award 07104.
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U.S. Public Health Service (USPHS)GM-11072
U.S. Public Health Service (USPHS)07104
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Caltech Gates and Crellin Laboratories of Chemistry6750
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Official Citation:Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy Timothy L. Legerton, Keiko Kanamori, Richard L. Weiss, and John D. Roberts Biochemistry 1983 22 (4), 899-903 DOI: 10.1021/bi00273a029
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ID Code:60756
Deposited On:05 Oct 2015 21:48
Last Modified:10 Nov 2021 22:38

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