Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development

Abstract

Postnatally the rat brain synthesizes catalytic forms of muscle type (MM) and heart type (MB) creatine kinase (CK), besides the supposedly sole type vertebrate brain-specific (BB) CK. We intended to demonstrate that in Rhode Island chicken brain, cytosolic (c) CK isoenzymatic transitions. (for example BB-CK is followed by the appearance of MB-CK and MM-CK during muscle differentiation), can also occur during development and aging. Cytosolic post 125000 x g, mitochondrial CK-free, brain samples were obtained for zone electrophoresis separation and identification of catalytically active cCK isoforms. BB-CK was never found during chicken brain ontogeny. Against the accepted view, an opposite isoenzyme transition pattern from MM through BB-CK was found in the chicken embryonic brain from the very early stages of development up to day 2 post-hatching. At very early stages of chicken brain ontogeny constitutive MM- and MB-CK isoenzymes were present before the advent of creatine. It seems to be that typical and atypical brain MM- and MB-CK could be working as ATPases in the absence of creatine before embryonic stage 28 (day 5.5) and/or such CK isoforms may begin to form part of the slow component b in developing early neurons and later in the nuclei of glial cells to be used by the CK/phosphocreatine (PC) system as the neural tissues mature. The post-hatching transition pattern showed simultaneous expression of more than one CK isoenzyme within the same neural sample as in post-natal rat brain, presumably due to regional differential transphosphorylation requirements. Strain-dependent enzymatic specific activities have been reported in several species. Since equivalent values of brain CK specific activity were obtained previously from the embryonic plateau phase of CK activity during White Leghorn development, and those from Rhode Island brain neurons cultured 11 days, we compared if, in vivo, a similar brain CK specific activity pattern was physiologically equivalent during Rhode Island and White Leghorn chicken ontogeny. We found quantitatively different strain-specific CK specific activity patterns during this period. Rhode Island brain CK activity values were approximately 4.5-fold those of White Leghorn ones. This indicates that production of energy from anaerobic metabolism and transphosphorylation by the CK/PC system to synthesize ATP more efficiently is strain-specific. In Rhode Islands, there was an age-dependent increase of CK specific activity, mostly in older animals (440% above the value found during the embryonic plateau), when the Krebs cycle and glycolysis lose capacity. During adult life and aging, under physiological conditions, the three CK isoenzymes may participate in diverse functions of the different cell compartments of brain glia and neurons with regard to their high and fluctuating energy demands that are not completely covered by anaerobic and aerobic glycolisis.

Evidence that 3‐hydroxyisobutyric acid inhibits key enzymes of energy metabolism in cerebral cortex of young rats

“…For total creatine kinase activity determination, the cerebral cortex was homogenized (1:1000, w/v) in isosmotic saline solution. For the preparation of mitochondrial and cytosolic fractions, the homogenates were centrifuged at 800 × g for 10 min at 4 °C and the pellet discarded (Ramirez and Jiménez, 2000). The supernatant was then centrifuged at 27,000 × g for 30 min at 4 °C in a Sorval DC‐2B centrifuge.…”

Evidence for a synergistic action of glutaric and 3‐hydroxyglutaric acids disturbing rat brain energy metabolism

“…For total creatine kinase activity determination, the cerebral cortex was homogenized (1:1000, w/v) in isosmotic saline solution. Mitochondrial and cytosolic fractions were prepared according to Ramirez and Jiménez (2000). Briefly, the homogenates were centrifuged at 800 × g for 10 min at 4 °C and the pellet (P1) discarded.…”

3-Methylcrotonylglycine Disrupts Mitochondrial Energy Homeostasis and Inhibits Synaptic Na+,K+-ATPase Activity in Brain of Young Rats

“…For total creatine kinase activity determination, the cerebral cortex was homogenized (1:10 w/v) in isosmotic saline solution. For preparation of mitochondrial and cytosolic fractions, the homogenates were centrifuged at 8009g for 10 min at 4°C and the pellet discarded (Ramirez and Jimenez 2000). The supernatant was then centrifuged at 27,0009g for 30 min at 4°C in a Sorval DC-2B centrifuge.…”