An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase.

All phosphagen kinases contain a conserved cysteine residue which has been shown by crystallographic studies, on both creatine kinase and arginine kinase, to be located in the active site. There are conflicting reports as to whether this cysteine is essential for catalysis. In this study we have used site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine and methionine. In addition, we have replaced Cys282, conserved across all creatine kinases, with alanine. No activity was found with the C282M mutant. The C282S mutant showed significant, albeit greatly reduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation) reactions. The K(m) for creatine was increased approximately 10-fold, but the K(m) for phosphocreatine was relatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reported for rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/K(creatine) profile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolate anion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determined spectrophotometrically and found to be 5.6 +/- 0.1. The S284A mutant was found to have reduced catalytic activity, as well as a 15-fold increase in K(m) for creatine. The pK(a) of Cys282 in this mutant was found to be 6.7 +/- 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing to the unusually low pK(a) of Cys282.

Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy.

Renal medullary interstitial cells are a prevalent and characteristic feature of the inner medulla of the kidney, but the physiological significance of this is unclear. We have developed a method for imaging renal medullary interstitial cells in situ by loading the cells with fluorescent dyes and monitoring their distribution using confocal microscopy. The pH-sensitive probe 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was used as a marker of cytoplasmic volume and therefore of cell morphology. Nile Red was used to demonstrate the presence of renal medullary interstitial cell lipid droplets. Papillae were excised from 100 g Sprague-Dawley rats and loaded with the appropriate dye. The papillae were then examined using a Leica TCS 4D confocal microscope and oil immersion lenses. Fluorescence was excited (488 nm) using an argon laser and emission wavelengths above 515 nm collected using a long pass filter. Images of papillae loaded with 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester clearly demonstrate a ladder-like arrangement of renal medullary interstitial cells. More detailed examination revealed the presence of cytoplasmic extensions that appear to make close contact with adjacent loops of Henle. Three-dimensional reconstructions of serial sections revealed spiral arrangements in some ladders of renal medullary interstitial cells. Nile Red-labelled lipid droplets of 0.5-1.0 microm diameter were located throughout the cytoplasm of renal medullary interstitial cells and especially within the cytoplasmic extensions. These experiments highlight the ability of confocal microscopy to allow investigation of renal medullary interstitial cells in situ.

Relationship between ATP resynthesis and calcium accumulation in the reperfused rat heart.

1. The postulate that the composition of solutions used to reperfuse ischaemic hearts may modulate their ability to synthesize high-energy compounds was tested in isolated rat hearts subjected to 30 min normothermic ischaemia and then reperfused with either Krebs'-Henseleit buffer (K-H) for 20 min (control reperfusion, CR), or a 'myocardial protective solution' (MPS) for 5 min, followed by 15 min K-H (modified reperfusion, MR). The 'myocardial protective solution' was designed to protect against damage caused by sodium and calcium accumulation and by free radicals. Metabolic precursors were also included to promote and support adenosine triphosphate (ATP) resynthesis during reperfusion under both aerobic and hypoxic conditions. 2. 31P nuclear magnetic resonance (NMR) was used to measure tissue ATP and creatine phosphate (CP), and atomic absorption spectrometry was used to measure Ca++. Early during CR, ATP recovered to 28% of the pre-ischaemic value, but fell to 5.5% with continued perfusion. Similarly, CP recovered to 45.5% of the pre-ischaemic level during early CR but fell to 25.5% with continued perfusion. 3. Better maintenance of ATP was seen during MR with oxygenated MPS (O2-MR), the final ATP remaining at 16.9% of the pre-ischaemic level. During O2-MR, CP recovered to 43.55 of the pre-ischaemic level but was not maintained and fell to a final level of 29.5%. 4. During MR with O2-free MPS (non-O2-MR), there was no reperfusion-associated fall in ATP or CP, with the levels maintained at 26.6% and 34.55, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Problems in the assessment of magnesium depletion in the rat by in vivo 31P NMR.

Prior in vitro studies, utilizing 31P nuclear magnetic resonance (31P NMR) to measure the chemical shift (sigma) of beta-ATP and lengthening of the phosphocreatine spin-spin (T2) relaxation time, suggested an assessment of their efficacy in measuring magnesium depletion in vivo. Dietary magnesium depletion (Mg2+ decreases) produced markedly lower magnesium in plasma (0.44 vs 1.13 mmol/liter) and bone (130 vs 190 mumol/g) but much smaller changes in muscle (41 vs 45 mumol/g, P less than 0.01), heart (42.5 vs 44.6 mumol/g), and brain (30 vs 32 mumol/g). NMR experiments in anesthetized rats in a Bruker 7-T vertical bore magnet showed that in Mg2+ decreases rats there was a significant change in brain beta-ATP shift (16.15 vs 16.03 ppm, P less than 0.05). These chemical shifts gave a calculated free [Mg2+] of 0.71 mM (control) and 0.48 mM (Mg2+ decreases). In muscle the change in beta-ATP shift was not significant (Mg2+ decreases 15.99 ppm, controls 15.96 ppm), corresponding to a calculated free Mg2+ of 0.83 and 0.95 mM, respectively. Phosphocreatine T2 (Carr-Purcell, spin-echo pulse sequence) was no different with Mg2+ decreases in muscle in vivo (surface coil) (Mg2+ decreases 136, control 142 ms) or in isolated perfused hearts (Helmholtz coil) (control 83, Mg2+ decreases 92 ms). 31P NMR is severely limited in its ability to detect dietary magnesium depletion in vivo. Measurement of beta-ATP shift in brain may allow studies of the effects of interaction in group studies but does not allow prediction of an individual magnesium status.