Isozyme Fractions Research Articles

#4346 SERUM NONSKELETAL ALKALINE PHOSPHATASE AS A MARKER OF METABOLIC ENDOTOXINEMIA AND PREDICTOR OF ADVERSE OUTCOME IN PATIENTS WITH CKD

Abstract Background and Aims Elevated alkaline phosphatase (ALP) levels are widely recognized as a marker of poor outcomes in chronic kidney disease (CKD). This association remains poorly understood, with arguments pointing to the skeletal and non-skeletal isozyme fractions as being decisive factors. The liver and intestinal isoenzymes of ALP are able to dephosphorylate and thus inactivate a number of substrates which have been implicated in the promotion of inflammation, including the bacterial endotoxin lipopolysaccharide (LPS). Present study aimed to test the hypothesis whether association of total ALP with all-cause mortality is mainly driven by non-skeletal ALP and its link with metabolic endotoxemia and inflammation. Method Laboratory parameters of mineral and bone disease, inflammation (C-reactive protein, CRP), and metabolic endotoxemia (Lipopolysaccharide binding protein, LBP) were assessed in 431 CKD patients enrolled in the Leuven Mild-to-Moderate CKD Study between Nov 2005 and Sept 2006. Non-skeletal ALP levels were estimated as residuals of the linear regression of log transformed total and bone-specific ALP. Spearman correlation, multivariate regression, and Cox proportional hazard analyses were performed to investigate the associations between skeletal and non-skeletal ALP, LBP, CRP and mortality. Data were censored at start of renal replacement therapy. Results Median age was 63 years, 54% were men, 19% had diabetes, 31% had established cardiovascular disease. At baseline, non-skeletal ALP was associated with inflammatory parameters (CRP: rho = 0.33, P<0.001) and with metabolic endotoxemia (LBP: rho = 0.32, P<0.001). Non-skeletal ALP was a significant predictor of elevated CRP (>3 mg/dL) in multivariate models (odds ratio (OR) per standard deviation (SD) increase 1.97; 95% CI 1.21-3.21; P = 0.006). This association was attenuated by further adjustment for LBP (OR 1.40; 95% CI 0.69-2.80; P = 0.3). During a median follow-up of 8.0 (interquartile range, 2.6-14.7) years 131 patients died. High non-skeletal ALP associated with increased risk of all-cause mortality, even after adjusting for demographics, Framingham risk factors and comorbidities (Hazard ratio (HR) per SD increase 1.28; 95% CI 1.05 – 1.56; P = 0.016). This association was attenuated by further adjustment for LBP (HR 1.22; 95% CI 1.00-1.50; P = 0.051). High bone-specific ALP was not associated with increased risk of all-cause mortality. Conclusion In patients with CKD not yet on dialysis, increased non-skeletal ALP (as opposed to skeletal ALP) is associated with inflammation and increased risk of all-cause mortality. These associations are at least partly driven by metabolic endotoxemia.

Impact of the first-generation drug-eluting stent implantation on periprocedural myocardial injury in patients with stable angina pectoris

Background and purposePercutaneous coronary intervention (PCI) with a drug-eluting stent (DES) is one of the standard treatments for patients with stable angina pectoris (AP). In spite of a notable effect in preventing restenosis after PCI, DES cannot improve the mortality of patients compared to a bare-metal stent (BMS). On the other hand, periprocedural myocardial injury (PMI) is related to poor prognosis in patients undergoing PCI. We compared DES to BMS in the incidence of PMI in patients with stable AP. Methods and subjectsWe enrolled 265 consecutive patients with AP undergoing successful stent implantation. A blood sample was obtained from all patients immediately before and 24h after PCI. PMI was defined as an increase in creatine kinase-myocardial band isozyme fraction (CK-MB) greater than the upper limit of reference range 24h after PCI. During the study period, sirolimus- and paclitaxel-eluting stents were used as DES. The strategy of PCI including the type of stent to implant was left to the discretion of the operator. ResultsPatients were divided into two groups (DES group, n=136 and BMS group, n=129). The incidence of PMI was significantly higher in the DES group than in the BMS group (24% vs. 12%, p=0.015). Use of DES remained an independent predictor of PMI on multivariate logistic regression analysis after adjustment for confounding factors (odds ratio 2.20, 95% CI, 1.07–4.51, p=0.032). ConclusionsImplantation of the first-generation DES including sirolimus- and paclitaxel-eluting stents was associated with a higher incidence of PMI in patients with AP compared to BMS.

Inactivation kinetics of food enzymes during ohmic heating

Ohmic heating of milk and fruit and vegetable juices was carried out at several incubation temperatures to investigate inactivation of alkaline phosphatase, pectin methylesterase and peroxidase. Mechanisms of inactivation of these enzymes and corresponding kinetic models were verified for each food material, using the multitemperature evaluation of inactivation data. Compared to inactivation by conventional indirect heating, kinetic parameters were changed but inactivation mechanisms remained the same. The kinetic parameter changes were relatively minor for pectin methylesterase and alkaline phosphatase. A significant destabilization of the labile isozyme fraction of peroxidase occurred by the effect of ohmic heating when the greatest decrease of stability was obtained for carrot juice.

Protein kinase A and signal transduction in T lymphocytes: biochemical and molecular methods.

Abnormal T-cell effector functions in systemic lupus erythematosus (SLE) are present and may be associated with disease immunopathogenesis. Our work has led to the characterization of a signaling defect, involving protein kinase A (PKA), leading to abnormal T-cell effector functions in SLE. PKA is a component of the adenylyl cyclase/cyclic adenosine monophosphate/PKA (AC/cAMP/PKA) pathway, a principal signal transduction system in T cells. The aim of this chapter is to provide a comprehensive, technical, step-by-step approach to studying PKA function in T cells. The methods detailed here are (a) chromatographic fractionation of PKA-I and PKA-II isozymes and PKA phosphotransferase activity in purified T cell populations, (b) Western immunoblotting to identify the presence of regulatory (R)-subunit proteins of PKA, and (c) isolation of RNA, and quantification of PKA R subunit-specific transcripts by competitive polymerase chain reaction. Although our emphasis in the chapter is T cells, these methods may be useful for investigation of signaling via PKA in other cell types as well.

Drought Stress, Peroxidase Activity and Formaldehyde Metabolism in Bean Plants

In our investigations we have studied the changes of total peroxidase and isozyme activities, peroxidase isozyme pattern and the level of endogenous formaldehyde and trigonelline. We have examined the effect of drought stress resulted by Carbowax treatment (2, 5, 7, 10%) of two different snap bean (Phaseolus vulgaris L.) genotypes [drought tolerant (T) and drought sensitive (S)]. There were no detectable differences in the peroxidase isozyme patterns but the total peroxidase activity was increased. We have found similar steep enhancement in the peroxidase activities of one of three peroxidase fractions while the increase in the other isozyme fractions was not so significant in the two genotypes. Increasing peroxidase activities and changes in the amount of endogenous formaldehyde and trigonelline were detectable with increasing Carbowax treatment in both genotypes, but in different ways. According to our experiments, the total peroxidase and some isozyme activity correlated with the concentration of endogenous formaldehyde and trigonelline, playing an important role during drought stress.

Purification and Characterization of Carboxylesterases of a Rice Green LeafhopperNephotettix cincticepsUhler

More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper,Nephotettix cincticepsUhler, could be resolved by isoelectric focusing electrophoresis. A combination of ammonium sulfate fractionation, gel filtration, and chromatofocusing chromatography was used to isolate and purify these isozymes. Four fractions, i.e., E1, E2, E3, and E4, with pI's ranging from 5.1 to 4.85, were obtained. The most abundant E3 had a molecular mass of 58.6 kDa and appeared electrophoretically homogeneous on SDS–PAGE. [1,3-3H]Diisopropyl fluorophosphate-labeling experiment revealed that the proteins of 58.6 kDa, a minor component of E2 and the major component of E4, were the carboxylesterase isozymes sought. A protein of the same molecular weight which existed in a very minute amount in E1 and was barely detectable on SDS–PAGE by Coomassie blue staining was actually the carboxylesterase isozyme of pI5.1. All four fractions exhibited significant activity toward several model substrates with α-naphthyl butyrate being the most preferred. Their activity toward malathion, permethrin, and cypermethrin was ca. 106-fold lower than their activity toward the model substrates. The pyrethroids were hydrolyzed more readily than malathion by these hydrolases, andcis-permethrin was more preferred than thetrans-isomer. E4 was the only fraction that cross-reacted with the antiserum against carboxylesterases of a rice brown planthopper,Nilaparvata lugens.Among the four isozyme fractions, E3, the most abundant, showed surprisingly low activity toward all four insecticides and was actually the least active fraction towardcis-permethrin and cypermethrin. A field strain ofN. cincticepshad 26- to 37-fold higher carboxylesterase activity toward the model substrates than a susceptible strain. Yet, little, if any, difference in the hydrolysis of malathion, permethrin, and cypermethrin was observed between these two strains. The field strain produced at least eight times more carboxylesterases than the susceptible strain.

Photoaffinity labeling of cottonseed microsomal N-acylphosphatidylethanolamine synthase protein with a substrate analogue, 12-[(4-azidosalicyl) amino]dodecanoic acid

N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), is synthesized from free fatty acids and PE in cotton seedlings (Chapman and Moore (1993) Plant Physiol. 102(3), 761–769). Here we use a photoreactive dodecanoic acid analogue, [12-(4-azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative to identify a protein subunit which corresponds to this cottonseed NAPE synthase activity. Dodecylmaltoside (DDM)-solubilized microsomal NAPE synthase enzyme was irreversibly and progressively inactivated by adding increasing concentrations of ASD and illuminating with UV 254 light. Protection from this photoinactivation was afforded by the natural substrate, palmitic acid. In low light, microsomal NAPE synthase utilized ASD as a substrate to synthesize NAPE; palmitic acid competed for this activity. NAPE synthase activity was measured directly in gel slices following nondenaturing PAGE of DDM-solubilized microsomal membrane proteins. Two-dimensional electrophoresis (nondenaturing PAGE, followed by SDS-PAGE) of photoaffinity-labeled, DDM-solubilized microsomal proteins revealed a 64 kDa polypeptide that was associated with the active NAPE synthase enzyme. Also, a 64 kDa protein was photoaffinity labeled in all NAPE synthase isozyme fractions isolated by preparative isoelectric focusing; photoaffinity labeling of this 64 kDa polypeptide was diminished in the presence of exogenously supplied palmitic acid. Collectively, our results demonstrate that ASD specifically interacts with NAPE synthase in a manner analagous to its fatty acid substrate and indicate that a 64 kDa polypeptide is a component of cottonseed microsomal NAPE synthase. ASD will be a useful molecular probe in future studies aimed at understanding the physiological role of this NAPE synthase enzyme in membranes of plant cells.

Isozymes of cottonseed microsomal N-acylphosphatidylethanolamine synthase: Detergent solubilization and electrophoretic separation of active enzymes with different properties

We recently reported that a novel acyltransferase activity (fatty acid : diacylphosphatidylethanolamine N-acyltransferase) synthesizes N-acylphosphatidylethanolamine (NAPE), an unusual derivative of phosphatidylethanolamine (PE), in microsomes of cotyledons of cotton seedlings by direct acylation of PE with free fatty acids (Chapman and Moore (1993) Plant Physiol. 102(3), 761–769). Here we report the solubilization of this membrane-bound NAPE synthase activity from cottonseed microsomes and the separation of three active isozymes with distinctly different characteristics. NAPE synthase activity was solubilized from NaCl-washed microsomal membranes by 0.2 mM dodecylmaltoside (DDM) at a 2:1 (w:w) detergent/protein ratio (assessed by enzyme activity after centrifugation at 150000 × g max, 1 h). Two sequential preparative isoelectric focussing separations of DDM-solubilized microsomal membrane proteins resulted in recovery of three distinct peaks of NAPE synthase activity - one at pH 6.3, one at pH 7.2, and one at pH 8.4 (designated A, B and C). These isozymes were purified 1148-fold (A), 269-fold (B), and 729-fold (C) from homogenates of cotton cotyledons. A 28 kDa subunit was enriched in all three isozyme fractions. Each of the isozymes exhibited unique kinetic properties with respect to palmitic acid and dioleoyl-PE. Each of the solubilized isozymes exhibited positive cooperativity toward palmitic acid (consistent with previous studies of NAPE synthase activity in intact microsomes) but not toward dioleyl-PE. Collectively, these results indicate that the synthesis of NAPE in cotton cotyledons is complex and has a potential for being a highly regulated process. The isolation of active NAPE synthase isozymes will provide the foundation for future studies into the physiological role of NAPE synthase (and NAPE) and the regulation of NAPE metabolism in membranes of plant cells.

ＡＬＰアイソザイム分析の現状と今後の展望 ＡＬＰアイソザイム染色法の問題点

ＡＬＰアイソザイム分析の現状と今後の展望 ＡＬＰアイソザイム染色法の問題点

血清ＡＬＰアイソザイムのセ・ア膜とＰＡＧディスク電気泳動法による成績の比較検討 消化器系疾患を中心に

血清ＡＬＰアイソザイムのセ・ア膜とＰＡＧディスク電気泳動法による成績の比較検討 消化器系疾患を中心に

Studies on human placental glutathione S-transferase. Multiplicity and mother age influence

Human placental glutathione S-transferase was purified to apparent homogeneity by direct application of the crude homogenate into glutathione linked sepharose affinity chromatography. Chromatofocusing analysis in the presence of reduced glutathione resolved the enzyme into three acidic peaks eluted at pH 6.0, 5.7 and 5.5. About 36% of the initial activity was recovered in the isozyme fraction eluted at pH 6.0 whereas the isozymes eluted at pH 5.7 and 5.5 accounted for 20% and 25% of the activity respectively. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a single protein band in all the three separated isozymes. These isozymes were homodimers with an apparent relative molecular mass of 44.000 and subunit molecular mass of 21.000. The isozymes were immunologically related to each other and to the enzyme from goat and sheep placentae. Mother age had no influence in the placental glutathione S-transferase activity, albeit the activity was slightly higher in placenta obtained from younger women.

Purification and characterization of chymotrypsin, trypsin and elastase like proteinases from cod ( Gadus morhua L.)

1. 1. Chymotrypsin, trypsin and elastase have been purified from the pyloric caeca of cod. 2. 2. The enzymes were separated by affinity/hydrophopic chromatography on phenyl-butyl-amine (PBA) substituted sepharose. Chymotrypsin eluted in two separate isozyme fractions whereas trypsin and elastase eluted in separate fractions consisting of two closely-related polypeptide chains as revealed by SDS-polyacrylamide electrophoresis and isoelectric focusing. 3. 3. The cod enzymes consist of single polypeptide chains with apparent molecular weights of about 27,000 Da as shown by denaturing polyacrylamide gel electrophoresis. 4. 4. The cod proteinases were retarded on gel filtration media. The retardation increased with increasing pressure. 5. 5. Isoelectric focusing analysis shows that the cod enzymes have isoelectric points in the range between 5 and 7. 6. 6. The cod proteinases are rapidly inactivated when stored at low pH's.

N-acetyl-beta-glucosaminidase in tissues of various human urogenital organs

The urinary level of N-acetyl-beta-D-glucosaminidase (NAG) was reported to be a useful marker for the assessment of renal function. This enzyme was also detected at a high activity in seminal fluid. In our preliminary report, seminal NAG activity was 1,000 times higher than urinary NAG level in healthy volunteers. Moreover, the fraction ratio A to B isozymes was changed in seminal fluid independently from that in urine. In order to study the source of seminal NAG, NAG activity and the isozyme fraction were determined in the tissues of the genital organs (prostate, testis, epididymis, and seminal vesicle) as well as the urinary organ (kidney). Various levels of NAG activities were obtained, the mean tissue concentration (Unit/g protein) being 26.7 +/- 2.4, 4.3 +/- 30.5, and 8.75 +/- 0.72 unit/g, respectively, in the testis, epididymis and seminal vesicle. In particular the mean concentration of 97.7 +/- 23.3 was significantly high in the prostate. In regard to the isozyme fraction of NAG, B fraction (%) accounted for 15.1 +/- 5.5, 33.6 +/- 8.2, 37.7 +/- 0.1, and 70.6 +/- 4.5 percent, respectively, in the testis, epididymis, seminal vesicle and prostate. It was to note that the A to B fraction ratio in the prostate corresponded to that in the seminal fluid. These results suggest that seminal NAG is mainly from the prostate.

Coenzyme Q10 and key enzyme activities in papillary muscle related to left ventricle function in mitral valve disease.

Coenzyme Q10 (CoQ10) was studied in papillary muscle from 18 patients (52-67 years, 2 females) subjected to open heart surgery due to mitral valve disease. In addition the enzyme activities of lactate dehydrogenase (LD) with its five isozymes, citrate synthase (CS) and mitochondrial CK (CK-MIT) were determined. Myocardial function was assessed by means of left ventricle (LV) angiography. CoQ10 averaged 0.39 (range 0.26-0.59) micrograms x mg-1 dw. On an individual basis CoQ10 was related to CS activity although not as closely as CK-MIT (r = 0.45, p less than 0.05 versus r = 0.86, p less than 0.001). The ratio (CoQ10) x (CS activity)-1 was calculated to represent mitochondrial quality. The level of LD3 fraction increase was used to mark for the degree of metabolic stress in the heart. LD3 fraction was negatively related to the quality index (r = -0.71, p less than 0.001). Thus, those with a low CoQ10 per unit of CS activity had also a high LD3 isozyme fraction. In a subset of 12 patients with isolated mitral regurgitation due to myxomatous valve degeneration, CoQ10 and the ratio CoQ10 over CS decreased with the degree of LV function impairment (r = -0.58, p less than 0.05 and r = -0.68, p less than 0.05, respectively). The quality index takes into account not only enzyme activity but also the potential for control of free oxygen radicals.

ChemInform Abstract: Enzymes in Organic Synthesis. Part 42. Investigation of the Effects of the Isozymal Composition of Pig Liver Esterase on Its Stereoselectivity in Preparative‐Scale Ester Hydrolyses of Asymmetric Synthetic Value.

AbstractCommercially available pig liver esterase (PLE) is separated in 6 different main isozyme fractions and it is shown that their individual stereospecifities are essentially the same in the hydrolysis of representative monocyclic and acyclic diester substrates (I), (III) (→ (II), (IV)).

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.

Maternal serum hexosaminidase A in pregnancy: effects of gestational age and fetal genotype.

The diagnostic usefulness of sulfated fluorogenic substrates in carrier detection of Tay-Sachs disease in serum during pregnancy was assessed by testing coded samples. Gradual increase in serum hexosaminidase activities toward these substrates was observed throughout pregnancy in both carrier and non-carriers of the Tay-Sachs gene, but absolute discrimination between the 2 genotypes could not be achieved even when values were compared within the same gestational age. Examination of isolated isozyme fractions with the sulfated substrates showed that the increased activities during pregnancy were due to a genuine increase in hexosaminidase A and not associated with the elevation of hexosaminidase I (or P), which was evident only with unsulfated substrates. The extent of the increase was influenced by the genotype of the fetus as indicated by higher values in pregnant carriers who carried non-carrier fetuses. We conclude that determination of serum hexosaminidase A during pregnancy by sulfated fluorogenic substrates may have a prenatal diagnostic value when used in obligate heterozygotes for Tay-Sachs disease, but is unreliable for screening purposes.

INCREASED SERUM HEXOSAMINIDASE A DURING PREGNANCY: IMPLICATIONS FOR TAY-SACHS CARRIER DETECTION BY SULFATED FLUOROGENIC SUBSTRATES

Carrier detection of Tay-Sachs disease (TSD) in sera is unreliable during pregnancy when based on the heat inactivation method. This is due to an increase in the relatively thermostable serum hexosaminidase (Hex) P, and is overcome by testing leukocytes. Since sulfated fluorogenic substrates (MUβGIcNAc-6-S & MUβGa1NAc-6-S) are highly specific for Hex A and provide direct determination independent of heat-labile/heat-stable ratio, we have examined their value in TSD carrier detection in pregnant women's sera. Determination of Hex activity toward sulfated substrates was carried out in isolated isozyme fractions from sera, leukocytes and fibroblasts of controls, pregnant TSD carriers and non-carriers, and TSD and Sandhoff disease patients. The results indicated that the cleavage en bloc of the N-acetylhexosamine-6-sulfate is catalyzed by an active site on the α-subunit, since only negligible activity was associated with Hex isozymes B, I1, I2 and P, and Hex S (α2) was twice as active as Hex A (αβaβb). The results also indicated that in addition to serum Hex P there is an increase in serum Hex A which starts during the second trimester of pregnancy. We conclude that accurate TSD carrier detection in serum during pregnancy may be possible if gestational age-matched controls are used.

A case of hypereosinophilic syndrome with LDH linked to IgG, A and M (κ, λ)-its sequential disappearance of Ig M, A and G by treatment

A 45-year-old female developed fever, erythema over the whole body, and swelling of general lymph nodes from mid-May 1984. At that time, the LDH was 929IU/l, each fraction was clear, and no anomaly was detected by immunoprecipitation reaction in free liquid media. The WBC in peripheral blood was 20, 900/μl, 18% of which were mature eosinophils. After administration of prednisolone at a daily dose of 30mg, the patient recovered and left the hospital. In February of the following year, fever and general erythema recurred. The WBC in peripheral blood was 31, 800/μl, of which mature eosinophils accounted for 42%. In sternal myelograph, mature eosinophils accounted for 9.2%. No whipworm eggs were found. A slight enlarged heart shadow and pericardial cavity fluid were noted, and a skin biopsy disclosed acanthosis and an infiltration of eosinophils around the blood vessels, but no vasculitis. The LDH in mid-March was 1, 182IU/l, and isozymes in each fraction were indistinct and LDH-linked immunoglobulins LDH-Ig G, A, M (κ, λ) were detected by immunoprecipitation reaction in free liquid media (molecular weight 520, 000). She was diagnosed of hypereosinophilic syndrome. From mid-March she was administered prednisolone at a daily dose of 40mg. The temperature declined gradually, the general erythema and pericardial cavity fluid subsuided, and the peripheral blood eosinophils also disappeared. The LDH became normalized in about October. At the end of March, the anomaly pattern was type 3-5, and LDH-Ig G, A (κ, λ) was detected. In mid-April, the anomaly pattern was 3-5, and LDH-Ig G (κ, λ) was observed. At the end of May, the anomary disappeared and not detected by immunoprecipitation reaction in free liquid media, but in the same serum after 2 weeks in cold storage, anomaly of type 3-5 appeared, and LDH-Ig G (κ, λ) was detected by immunoprecipitation reaction in free media. At the end of May 1986, the LDH was 231IU/l, the isozyme fractions were clear, and no anomaly was detected by immunoprecipitation reaction in free liquid media, nor was it detected in the same serum after 2 weeks in cold storage. This case presents three interesting points in considering the onset mechanism of LDH-Ig. 1) At the time of the first admission, the LDH was high, but no anomaly was noted. At the time of the second admission, when the LDH increased again, an anomaly occurred. 2) As a results of treatment, LDH-IgM, IgA, IgG disappeared in this sequence. 3) No anomaly was noted in the serum sampled during the process, but an anomaly was found in same serum after 2 weeks in cold storage.

市販β‐ＮＡＤ｀＋´標品中不純物質によるヒトＬＤＨアイソザイムのサブバンド形式

When human lactate dehydrogenase, LDH (L-lactate: β-NAD+ oxidoreductase, EC 1. 1.1.27) was incubated with β-NAD+, five bands consisting of original band and four subbands were formed in each isozyme fraction of LDH1(B4), LDH2(B3A), LDH3(B2A2), LDH4(BA3), and LDH5(A4), and at the same time LDH activity of the reaction mixture decreased with increase in the subband formation.The subbands formed differed in their number, mobility, and the ratio of each band considerably depending on the commercial NAD+ samples used. The decrement of the activity also depended on them. These two characteristic changes also depended a great deal on the kind and pH of the buffers used.The result obtained led us to conclude that the changes mentioned above were produced by some interfering substance contaminated in the NAD+ samples which bound each subunit in LDH tetramer in one to one ratio, maximally binding all of the four subunits.