Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.     

Isoenzyme pattern of serum beta-hexosaminidase in liver disease, alcohol intoxication, and pregnancy

The isoenzyme distribution of serum beta-hexosaminidase was studied in 11 pregnant women, 18 patients with cirrhosis of the liver, 14 patients with cholestasis, and 9 chronic alcoholics with acute ethanol intoxication. Serum beta-hexosaminidase activity was elevated in sera from all groups. The results of isoenzyme separation by isoelectric focusing demonstrated that the isoenzyme pattern in the different patient groups was similar to pregnancy with a preferential increase of isoenzyme forms with pI's between 5.6 and 6.8.     

Secretion of beta-hexosaminidase by cultured human skin fibroblasts. Kinetics, effect of temperature, cell density, serum concentration and pH.

The temporal relation between the fusion of mononucleated myoblasts into multinucleated fibres and the quantitative changes in the activity of creatine kinase isoenzymes was determined in rat skeletal muscle cell cultures. The effect of actinomycin D on the isoenzyme transition was investigated. The activity of creatine kinase in cultures prior to the onset of cell fusion is predominantly of the BB type. During the phase of cell fusion, there is a manyfold increase in creatine kinase activity. This is due to the appearance or great increase in the activity of the MM isoenzyme. During this period, increase in the BB isoenzyme activity is very small. Inhibition of RNA synthesis by actinomycin D shortly before the onset of cell fusion did not prevent cell fusion and isoenzyme transition during the first 6 h following application of the drug.     

The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis: purification, identification of a heterodimer and differential heat sensitivity

The three Cu,Zn superoxide dismutase electromorphs of the amphibian Xenopus laevis were purified by an original procedure. N-terminal sequence analysis demonstrated that they are two different homodimers (AA and BB) and a hybrid heterodimer (AB), arising from the co-expression of duplicated genes. The three forms have the same pI, same enzyme activity and EPR spectra, but different heat-sensitivity, form BB being more resistant than form AA, with form AB showing intermediate sensitivity. Thermostability of BB and the control bovine enzyme was enhanced by a tenfold increase in protein concentration. It is suggested that the higher heat sensitivity of the AA isoenzyme is related to the presence of an extra Cys residue and to an easier dissociation of the protein dimer into monomers.     

Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I

UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoenzyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.     

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, -dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.     

Changes in the activity of adenylate kinase isoenzymes in the ischemic rabbit heart

Under the influence of 1 hour myocardial ischemia activity of rabbit heart mitochondrial isoenzyme AK2 increased by 40%, but the activity of matrix isoenzyme AK3 decreased by 77%. No changes were found both in total adenylate kinase activity, and cytosolic isoenzyme AK1. The reasons of these alterations are not sufficiently clear. Apparently, they are related with functioning conditions of these isoenzymes in ischemic tissue.     

Change in cell membrane permeability, and composition and properties of hexokinase during induced carcinogenesis]

The isoenzyme hexokinase (HK) spectrum from normal rat large intestinal mucosa consisted of 3 isoenzymes. In tumours of this localization induced by 1,2-dimethylhydrazine there proved to be a lack or marked decrease in the most rapid anodic isoenzyme. Only one HK isoenzyme was found in the metastases. Km (glucose) for tumour HK was 2--3 times lower than for normal intestinal HK; the HK activity was detected in the serum from the 1st month of the carcinogenic administration, and by the 5th month it was found in 80% of the tumour-bearing animals. No serum HK activity was ever found in control rats.     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.     

Heat-stable alkaline phosphatase in uterine cancer, with special reference to its histochemical heat-stability and the L-phenylalanine inhibition test

Summary We report a histochemical study of alkaline phosphatase (ALP) in normal cells of the female reproductive system, in pre-cancerous and cancerous lesions of the uterine cervix and in endometrial cancer to ascertain the incidence of ALP and its isoenzyme type. For this purpose, serial sections were subjected to heat stability andl-phenylalanine (LP) inhibition tests.The Regan-like isoenzyme, a heat-stable and LP-sensitive ALP, which has been thought to derive only from cancer or the placenta, was found in uterine cervical reserve cells and endometrial luminal surface lining cells. In contrast, ALP activity in endometrial glandular cells was found to be heat and LP sensitive.Of 183 cases of cervical neoplasia, 60 (33%) manifested non-specific ALP activity. One dysplasia and two invasive cancer cases manifested the Regan-like isoenzyme. The other 36 classifiable lesions had small-intestine ALP-like activity (marked heat and LP sensitivity) or a liver ALP-like isoenzyme (marked heat and slight LP sensitivity).Of 42 cases of endometrial cancer, all cases manifested non-specific ALP activity. Seven endometrial cancers exhibited the Regan-like isoenzyme. The other 19 cases manifested either small intestine of liver ALP-like isoenzyme.Our findings indicate that in the course of uterine carinogenesis, the ALP isoenzyme of reserve cell and endometrial glandular cells undergo a change and that enzyme deviation occurs.     

Characteristics and Diversity of beta-d-Glucosidase (EC 3.2.1.21) Activity in Marine Snow

Large amorphous aggregates commonly described as marine snow were sampled in the water column of the northern Adriatic Sea in August 1991. beta-d-Glucosidase activity associated with these aggregates was characterized. Enzymatic activity was measured with the fluorogenic substrate analog 4-methylumbelliferyl-beta-d-glucoside and found to be mainly associated with particles; on average, only 24% of the whole beta-d-glucosidase activity remained in the supernatant after centrifugation. Although the temperature optimum for beta-d-glucosidase activity was approximately 40 degrees C, incubation of the previously liberated particle-attached enzyme at 50 degrees C for 20 min caused a >90% reduction of enzymatic activity relative to the activity at 40 degrees C. The level of inactivation of beta-d-glucosidase was much lower, however, when whole marine snow was incubated, indicating qualitative modifications of beta-d-glucosidase in marine snow. Separation of beta-d-glucosidase by different approaches indicated that the diversity of isoenzymes is restricted. In samples taken from the pycnocline, only one major isoenzyme was present in noticeable amounts. This isoenzyme contributed up to 70% of the whole beta-d-glucosidase activity detectable by two different chromatographic separations (anion-exchange chromatography and size exclusion chromatography). Although the same isoenzyme was dominant in marine snow taken from surface waters (0.5-m depth), we found a second peak of activity which eluted at lower NaCl concentrations from the anion-exchange column. Generally, the diversity of isoenzymes exhibiting beta-d-glucosidase activity seems to be surprisingly small in amorphous pelagic aggregates.     

Purification and partial characterization of high and low activity carbonic anhydrase isoenzymes from Malaclemys terrapin centrata.

1. High activity (CA C) and low activity (CA B) carbonic anhydrase isoenzymes have been purified from turtle erythrocytes. 2. The two isoenzymes differed in CO2 hydration specific activity by 36-fold. 3. The low activity isoenzyme contained one half-cystine residue, whereas the high activity isoenzyme contained four half-cystines and required a reducing environment to maintain activity. Both isoenzymes contained zinc. 4. Molecular weights of 28,500 and 30,400 daltons were established for the low and high activity isoenzymes respectively. 5. Both isoenzymes were inhibited by acetazolamide, but only the high activity isoenzyme was inhibited by parachloromercuribenzoate. 6. The low activity isoenzyme was present in the erythrocytes at about 8-10 times the concentration of the high activity isoenzyme. 7. The high activity isoenzyme cross-reacted with antibodies prepared against pure chicken carbonic anhydrase C.     

Ontogenetic Development of Creatine Phosphokinase in Skeletal Muscles and Heart from Pigs

Creatine Phosphokinase (CPK) in striated muscles shows only small changes in activity before birth. After birth and during the first month of extrauterine life the activity increases rapidly. The largest increase is seen in muscles with a glycolytic energy metabolism (m. long, dorsi) and the smallest in muscles with an oxydative energy metabolism (m. flexor dig. ped. sup.). The differences between these groups of muscles are statistically significant. In heart tissue the increase in CPK activity is lower, the levels amounting to 40 to 47 % of those in striated muscles. Early in fetal life only the BB isoenzyme is found in striated muscles. Synthesis of M subunits of GPK starts between day 76 and 65 before birth and increases rapidly after this time leading to disappearance of the BB isoenzyme 24 days prior to birth and of the MB isoenzyme at birth. In muscles with an oxydative as well as in muscles with a glycolytic metabolism all GPK activity after birth is caused by the MM isoenzyme. All three isoenzymes are present in heart tissue at the earliest prenatal stage investigated, the pattern being dominated by the BB isoenzyme. During further differentiation the MM isoenzyme increases and the BB isoenzyme decreases. The development is completed during the first month after birth with a final isoenzyme composition of 81 % MM and 19 % MB isoenzyme. kw|Keywords|k]pigs; k]ontogenesis; k]creatine phosphokinase; k]activity; k]isoenzymes     

LDH and LDH Isoenzymes of Synovial Fluid in the Horse

LDH is an intracellular enzyme, which when cells degenerate is released to the extracellular spaces and body fluids. Cells and organs in the mammalian body differ from each other with respect to their LDH isoenzyme patterns. These circumstances have led to the use of LDH isoenzyme determinations in laboratory diagnostic work. In the present investigation total LDH activity and LDH isoenzyme distribution in equine synovial fluid from healthy joints, joints with serous arthritis, osteochondrosis dissecans and arthrosis, were determined. The fluids from the diseased joints differed from normal synovial fluid with respect to total LDH activity, and the different joint diseases each seemed to give rise to a characteristic isoenzyme pattern. In order to examine possible sources of the increased LDH activity and altered isoenzyme patterns, blood plasma, red and white blood cells, synovial membrane and articular cartilage were also studied. It was found that LDH4 and LDH5 were present in high amounts in articular cartilage, and an increase in these isoenzymes was the most characteristic feature in synovial fluid from joints with arthrosis. The results were discussed in view of possible diagnostic value of isoenzyme determinations on synovial fluid.     

Isolation, purification, and characterization of thermophilic T80 isoenzyme of xylose isomerase from the xerophyte Cereus pterogonus

A thermostable isoenzyme (T(80)) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K(m) and V(max)) of the T(80) xylose isomerase isoenzyme were investigated. The molecular mass of the purified T(80) isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T(80) isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84?KJ mol(-1). The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T(80) isoenzymes yielded an emission peak with λ(em) at 320?nm and 340?nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn(2+) and Co(2+) associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production.     

离子注入对萝卜过氧化物酶、淀粉酶和蛋白酶同工酶的影响

研究了氮离子和氩离子注入萝卜种子对萝卜幼苗蛋白含量、过氧化物酶活性及过氧化物酶、淀粉酶和蛋白酶同工酶的影响。结果表明 :离子注入后 ,减低萝卜过氧化物酶活性和蛋白含量。萝卜不同生长时期同工酶变化不一样。在子叶时期 ,过氧化物酶同工酶谱带无明显变化 ,淀粉酶同工酶有酶带的消失 ;而在真叶时期 ,过氧化物酶在负极区减少一条酶带 ,Rf为 0 .2 2 ,正极区增加一条酶带 ,Rf为 0 .6 ,且随剂量增加 ,酶带着色增强 ;淀粉酶同工酶在注入剂量为 5× 10 5N+ / cm2 )时 ,有同工酶带增加 ,Rf为 0 .6 1。低剂量时蛋白酶活性增强 ,谱带增多 ,大剂量则减弱。因此 ,N+和 Ar+注入后 ,可影响萝卜过氧化物酶和淀粉酶的表达及蛋白质的合成或降解。     

Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.     

Comparative study of alkaline phosphatase isoenzymes,bone histology,and skeletal radiography in dialysis bone disease.

Liver, intestinal, and bone alkaline phosphatase isoenzymes were measured using heat stability and L-phenylalanine inhibition techniques in 78 patients on intermittent haemodialysis. Fifty-five patients had abnormalities in one or more of the isoenzymes. Changes in bone and intestinal alkaline phosphatase activities seemed to be related and raised liver isoenzyme activity was associated with the development of liver disease. Abnormal histological and radiological findings were better correlated with bone alkaline phosphatase levels than with total alkaline phosphatase, and serial estimations of bone isoenzyme activity were useful in assessing the response of renal osteodystrophy to treatment with a vitamin D analogue. Serum alkaline phosphatase isoenzyme measurement provides another useful and non-invasive index for monitoring metabolic bone disease in patients with chronic renal failure.     

LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax

The lactate dehydrogenase isoenzyme patterns have been studied in the axial muscles of the sharks Etmopterus and Galeus. Samples from red, intermediate and white muscle fibres were run separately on a polyacrylamide slab-gel. Both sharks have three isoenzymes; all three are present in the red and intermediate fibres, while the white fibres contain only the two slowest-moving isoenzymes. The red fibres of both sharks contain most of the fastest-moving isoenzyme.
The isoenzymes have a high tolerance towards urea; the slow moving isoenzyme is inhibited at about 2 m urea, the next isoenzyme at 4-6 M urea, and some activity of the fast-moving isoenzyme is still present at 10 M urea in the incubation medium. The LDH distribution in the fibre types is studied by histochemistry on frozen sections.     

Ginkgo biloba extract (EGb 761) protects Na,K-ATPase isoenzymes during cerebral ischemia.

Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.