Jozef Orlický

Alanine Aminotransferase in Bovine Brain: Purification and Properties

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140‐ and 180‐fold, respectively) from bovine brain cortex by means of (NH4)2SO4 precipitation, gel filtration on Sephadex G‐150, and ion‐exchange chromatography on DEAE A‐50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C‐50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The Km values of the mitochondrial enzyme were 5.1 mM, 6.6 mM, 0.7 mM, and 0.4 mM and of the cytosolic isozyme were 30.3 mM, 4.3 mM, 0.7 mM, and 0.5 mM for alanine, glutamate, 2‐oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.

Introduction to a kinetics-sensitive double-step voltcoulometry

As an alternative to the numerous state-of-the-art versions of voltammetry, a kinetics-sensitive double-step voltcoulometry is introduced. The transient current flowing in response to a potential step across the electrochemical cell is integrated and simultaneously processed by a deliberately selected time-domain “cascade” filter, while scanning the applied potential. In contrast to the widely used sampling scheme of sampling the transient current just before and in the end of the excitation pulse, three values of the transient charge are sampled in the interval between subsequent excitation pulses. Each measuring period is preceded by a single measurement of the steady-state current with the excitation pulse being switched off. The latter measurement makes it possible to actively compensate the parasitic charge across the feedback capacitor of the integrator, due to the steady-state current, while storing the steady-state current data. The goal of introducing the third sampling event resides in discrimin...

Exemplifying performance of kinetics-sensitive double-step voltcoulometry : redox reactions of protons in unsupported acids

Abstract An alternative electroanalytical technique to the widely used differential pulse voltammetry (DPV) is presented in connection with monitoring proton redox reactions in strong acids (HCl, H2SO4, HNO3) and in l -ascorbic acid (AA) in the absence of any supporting electrolyte. Contrary to the DPV method, the current flowing through the working electrode in response to a double-step change of the applied potential is first integrated and subsequently processed by a three-channel correlator. Expressing the faradaic transient charge as Q(t)∝tβ, the ratio Rβ=[Q(t1)−2Q(5t1)+Q(9t1)/(Ilimt1), where t1 is the delay of the first sampling event with respect to the trailing edge of the potential double step and Ilim is the limiting current of the corresponding steady-state voltammetric wave, it is calculated and then compared to the values found experimentally. The sensitivity to the kinetics represented as dRβ/dβ has an optimum around β=0.5, a value consistent with the Cottrell equation. The experimental data point to a crucial role of CO2 (H2CO3) dissolved in the acid solution, envisaged as the reversed sign of the measured charge. After deaerating the solution by argon the sign became positive, nevertheless the experimental Rβ values were systematically higher than the predicted ones. The reaction of the protons of AA at negative potentials seems to be of the EC type when proceeding from negative to more positive potentials. Moreover, there is a dominant voltammetric wave of AA at positive potentials coming from an irreversible reaction, accompanied by a relatively weak peak of the correlated charge.

Isoelectric focusing of the alanine aminotransferase isoenzymes from the brain, liver and kidney

1. The isoelectric points of mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) of bovine brain cortex, of rat, guinea-pig and human livers and of rat and pig kidney cortices were estimated. 2. The pI of the cytosolic enzymes were found at about 5.2 and those of mitochondrial ones at 7.2. 3. It is suggested that in the examined tissues except for the human liver there are two different proteins with the alanine aminotransferase activity.

Alanine aminotransferase and lactate dehydrogenase activity in the crayfish and rabbit striated muscles.

Abstract 1. 1. The alanine aminotransferase. (E.C.2.6.1.2) and lactate dehydrogenase, (E.C.1.1.1.27.) activities were determined in the crayfish and rabbit striated muscles. In the crayfish muscle the alanine aminotransferase activity was of an order higher than that in the rabbit one, while the lactate dehydrogenase activity was absolutely higher in the rabbit muscle. 2. 2. Alanine aminotransferase activity was present in both species mainly in the cytosolic fraction. Enzyme from both animal species had the same pH optimum, (between 7.6 and 8.2) K m values and the relative increase of the activity depending on the temperature. 3. 3. The reagents blocking free SH and carbonyl groups of proteins as well as antimetabolite of B 6 -vitamin Penicillamine inhibited the alamine aminotransferase activity in both animal species to the same extent. 4. 4. A different metabolic role in the metabolism of crayfish and rabbit muscles is suggested.

Alanine formation and alanine aminotransferase activity in the nerve tissue with proliferating macroglia.

SummaryIn the nerve tissue with proliferating macroglia cells were observed a lowered oxygen consumption, an increased aerobic glycolysis and alanine formation and a higher alanine aminotransferase and glutamate dehydrogenase activity than in the control tissue in the homogenates and in the cell sap fraction. The substrate saturation curves, apparent Km and pH optimum values in the tissue with proliferating macroglia and in the control did not differ from one another. The authors assume that a higher alanine amino-transferase activity in the tissue with macroglia proliferation can reflect either a higher synthesis of the enzyme in the altered tissue, or a predominance of glial elements in the altered tissue possessing a higher alanine aminotransferase activity than the nerve cells.