Biomedical subjects
D A Nealon
Publications and source records attributed to D A Nealon.
Isoelectric focusing of the cytosolic human creatine kinase isoenzymes.
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Quantitation of aspartate aminotransferase isoenzymes after electrophoretic separation.
A scheme for the quantitative detection of aspartate aminotransferase isoenzymes and multiple forms after electrophoretic separation is described. Glutamate generated from the aminotransferase reaction is quantitated by using the glutamate dehydrogenase/diaphorase-coupled enzyme system to form a formazan dye. Product inhibition of aspartate aminotransferase by oxaloacetate is prevented by including oxaloacetate decarboxylase in the overlay reagent. Results compare favorably with those of an immunochemical precipitation procedure. The method can also be used to detect quantitatively subforms and atypical forms (genetic variants, immunoglobulin-enzyme complexes) of aspartate aminotransferase.
Rapid purification of adenylate kinase from human erythrocytes and skeletal muscle.
Adenylate kinase from human erythrocytes and skeletal muscle can be purified to homogeneity by a new procedure based on DEAE-Sepharose and Blue Sepharose affinity chromatography and Sephadex G-75 fractionation. For the enzyme purified from erythrocytes the specific activity is 3,000 U/mg of protein, and the overall yield is 70%. For the enzyme purified from skeletal muscle the specific activity is 2,075 U/mg of protein, and the overall yield is 44%. The sequence of steps takes advantage of the high isoelectric point, the high affinity for Blue Sepharose, and the low molecular weight of the isoenzyme from these two human tissues.
Naming of creatine kinase subtypes.
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Relative inhibition of human adenylate kinase and creatine kinase isoenzymes by adenosine 5'-monophosphate and diadenosine pentaphosphate.
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Effects of temperature on measurement of alkaline phosphatase activity.
We examined the effects of temperature on the activity and steady-state kinetic properties of alkaline phosphatase (EC 3.1.3.1). Purified isoenzymes from human liver, intestine, and placenta were used, as was human serum, and the enzyme from porcine kidney. Phosphatase activity was estimated by two different assay techniques. For all isoenzymes, apparent Michaelis constants for the substrate 4-nitrophenyl phosphate decreased with increased temperature; Km at 37 degrees C was typically half that determined at 25 degrees C. All enzymes of human origin exhibited similar linear Arrhenius relationships over the range examined, 20-37 degrees C (Ea of 30-36 kJ X mol-1). The porcine kidney enzyme obeyed an Arrhenius relationship that was slightly, but significantly, different from the isoenzymes of human origin. Temperature relationships based upon Arrhenius behavior and individual activity measurements are presented. For human alkaline phosphatases, they differed by no more than 10%.
Creatine phosphate does not inhibit human adenylate kinases.
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Enzyme measurements by mass: an interim review of the clinical efficacy of some mass measurements of prostatic acid phosphatase and isoenzymes of creatine kinase.
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Activation of human creatine kinase isoenzymes by pH and various sulfhydryl and chelating agents.
We report the effect of serum pH and of the presence or absence of mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate on the activation of the human creatine kinase isoenzymes. At the serum pH giving maximal enzyme stability and minimal assay lag phase (Nealon et al., Clin. Chem. 26: 1165-1169, 1980) thiol activation of CK-1 and CK-3 is nearly maximal with monothioglycerol in an optimized creatine kinase assay (Szasz et al., Clin. Chem. 22: 650-656, 1976). However, CK-2 is maximally activated at pH 8.5, a pH at which this isoenzyme is least stable on storage and its assay lag phase is prolonged. These findings suggest irreconcilable problems in the storage, activation, and assay of CK-2.
Purification of lactate dehydrogenase isoenzyme-5 from human liver.
We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).
Diluent pH and the stability of the thiol group in monothioglycerol, N-acetyl-L-cysteine, and 2-mercaptoethanol.
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A rapid and simple procedure for the preparation of human lactate dehydrogenase-2 from erythrocytes using an ion-exchange mini-column.
We describe a simple ion-exchange chromatography technique for the rapid preparation of lactate dehydrogenase-2 from lysed human erythrocytes obtained either from plasma or serum clots. The procedure appears capable of scale-up to larger columns thus making it ideal as an initial step in a more extensive purification scheme.
Creatine kinase-1 is principally inactivated in serum by complexing with immunoglobulin-G.
1. The stability of enzyme activity of the creatinine kinase-1-immunoglobulin-G complex has been determined at 37 degrees C, 4 degrees C and -20 degrees C in heat-inactivated serum and buffer. 2. The complex was formed by incubating creatinine kinase-1 and immunoglobulin G at 37 degrees C for 30 min. It was isolated by Sephadex G-100 column chromatography. 3. At all temperatures the complex suspended in buffer was about twice as stable as it was in heat-inactivated serum. Stability decreased in the sequence of 4 degrees C, 37 degrees C and -20 degrees C. Therefore all isolations of the complex were carried out at 4 degrees C. 4. At 37 degrees C the decay of enzyme activity of the complex was found to be biphasic first order. In serum the Kd values were -0.045 min-1 and -0.0028 min-1, and in buffer -0.023 min-1 and -0.0016 min-1. 5. From column-chromatography experiments it was found that between 10 and 20% of the total creatine kinase-1 was involved in the complexing reaction after a 30-min incubation. 6. With this 10-20% proportion it can be calculated that the overall t0.5 for the decay of total creatine kinase-1 activity must be in the range 95-201 min. This finding suggests, by comparison with other published data, that the enzyme-immunoglobulin complex is the main route of creatine kinase-1 catabolism in serum. 7. The difference between serum and buffer decay values for the complex is possibly due to the presence of cystine, urate and other substances in serum, which are additional potent creatine kinase inhibitors.
Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes.
We report the effect of serum pH on the storage stability of the human creatine kinase isoenzymes and on the creatine kinase assay lag phase (Szasz et al., Clin. Chem. 22: 650, 1976). We also investigated the effect of including mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, or ethylene glycol bis(betaaminoethyl ether)-N,N,N',N'-tetraacetate at 20, 4, and --20 degrees C. Storage stability of the isoenzymes is profoundly affected by pH. For patients' samples and semi-purified human creatine kinase isoenzymes added to heat-inactivated sera, increasing diluent pH above 7.0 decreases creatine kinase stability. The thiol agents or chelators generally give little or no protection above pH 7.5; at pH 8.5 they contribute significantly to isoenzyme instability. Storage at 4 degrees C provides greater stability than storage at 20 degrees C, particularly in the case of creatine kinase isoenzyme BB. The lag phase was minimum at a serum pH of 6.5, in the presence of 10 mmol of monothioglycerol per liter. Increasing serum pH to 8.5 prolongs the reaction lag phase by about 1 min over the minimum. We recommend that, before they are stored at 4 degrees C, the pH of patients' samples be adjusted to 6.5 and oxidation of SH-groups be minimized by adding monothioglycerol to the sample.
[Bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane is an effective buffer for creatine kinase assays.
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Effect of cations on the human creatine kinase isoenzymes.
We report our findings on the effect of Ca2+, K+, Na+, Fe2+, Mn2+, Zn2+, and Cu2+ on the activity of creatine kinase isoenzymes derived from human tissues. The only inhibitory cation was Ca2+, and Dixon plots of the data indicate the inhibition to be competitive for the Mg2+ in the reaction assay mixture. This effect of Ca2+ on all three isoenzymes is completely reversed by including Mg2+ (10 mmol/L) and ethylenediaminetetraacetate (2 mmol/L) in the assay mixture. Our findings suggest that it is unnecessary to add chelators to the patient's sample.
Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa.
Neurospora crassa nicotinamide adenine dinucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis and sedimentation equilibrium. Purification of the enzyme is facilitated by the presence of phenylmethanesulfonyl fluoride and by the use of a ribose-linked adenosine 5'-monophosphate affinity column. The enzyme appears to be composed of nonidentical subunits of molecular weights 42 800 and 38 300 as estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate. From the intensity of each band and the native molecular weight, it is concluded that the enzyme is composed of either six or eight subunits, three or four of each type, respectively. The availability of pure enzyme will allow clarification of the structure of the enzyme by ligand binding studies.