K Elmdén | PubMed Health

PubMed1988

Evidence against major compartmentalization of H+ in ischemic rat brain tissue.

Ischemia leads to intracellular acidosis, the severity of which depends on the availability of glucose for production of lactate and H+. It has been suggested that major compartmentalization of H+ occurs, with glial cells becoming much more acidotic than neurons. Since this issue is of crucial importance for the understanding of mechanisms of ischemic brain damage, we induced complete ischemia by decapitation in hypo-, normo- and hyperglycemic awake rats, yielding brain tissue lactate contents varying between 4 and 27 mumol/g. Using phosphorus nuclear magnetic resonance (NMR) we explored whether a splitting of the phosphorus peak occurred as a reflection of compartmentalization. Forebrains were put in NMR tubes and spectra obtained 15 min after decapitation. Since no such splitting was observed, we conclude that major compartmentalization does not occur in ischemia at the degrees of lactic acidosis studied.

Animals↗

PubMed1987

Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D9k.

Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.

Aminoquinolines↗