In order to determine the enzyme activity, as expressed in Reitman-Frankel unit, of GOT isozyme present in whole homogenate, mitochondrial fraction and supernatant fraction were prepared from brain tissues of normal adult rabbit, by a differential centrifugal method. The effect of thiopental on the GOT isozyme activity in each fraction was determined and the following results were obtained. 1) The activity of GOT isozyme in whole homogenate of normal rabbit brain tissues was found to be 545+/-2.608 units/mg of wet weight whereas the corresponding figure for the supernatant GOT isozyme was 512+/-3.081 and the value for the mitochondrial GOT isozyme was found to be 34.9+/-1.224. 2) The supernatant GOT isozyme existing in a floating status within the cytoplasm accounted for 94 percent followed by 6.35 percent of mitochondrial GOT isozyme. 3) The activated-peak of mitochondrial GOT isozyme contained in the whole homogenate of adult rabbit brain tissues was found to be at #15 on the tube of elution in comparison to that of #73 for supernatant GOT isozyme, as analyzed by the DEAE-Cellulose column chromasography. 4) The supernant GOT isozyme from the thiopentaltreated brain was proportionaly distorted while mitochondrial GOT isozyme was not influenced. Fro example, treated with thiopental, the supernatant GOT isozyme was divided to be #63 & #73 on the tube in comparison to #15 for the mitochondrial GOT isozyme. 5) The activity of supernatant isozyme was proportionaly reduced as the concentration of thiopental. 6) Fifty percent inhibition dose(1se) of thiopental on the supernatant GOT isozyme was found to be 0.63mM. 7) The inhibitory effect of thiopental on the supernatant GOT isozyme was very high significantly by the statistics. 8) The mchanism by which thiopental inhibits the supernatant GOT isozyme in the adult rabbit brain was found to bh uncompetitive inhibition as its Michaelis-Menten constant Km=58.07mM demonstrated. In view of the above finding it is suggested that the thiopental inhibited selectively the activity of supernatant GOT isozyme of the adult rabbit brain tissues while it did not inhibitnificantly by the statistics. 8) The mechanism by which thiopental inhibits the supernatant GOT isozyme in the adult rabbit brain was found to bh uncompetitive inhibition as it Michaelis-Menten constant of Km=58.07 mM demonstrated. In view of the above findings it is suggested that the thiopental inhibited selectively the activity of supernatant GOT isozyme of the adult rabbit brain tissues while it did not inhibit that of mitochondrial GOT isozyme. The GOT isozyme of adult rabbit brain tissues was divided into thiopental-sensitive GOT isozyme(supernatant GOT isozyme) and thiopental insensitive GOT isozyme(mitochondrial GOT isozyme) Furthermore, it is suggested that the cellular function of the brain can be somewhat hindered, when thiopental is injected into the brain cell, while mitosis of the brain cell is not influenced.
Adult* ; Aspartate Aminotransferases* ; Brain* ; Cytoplasm ; DEAE-Cellulose ; Humans ; Mitosis ; Thiopental*

OBJECTIVETo optimize extraction and purification methods of acidic polysaccharide from Moerella iridescens (MIAP).

METHODSWith alkali extraction process and orthogonal experiment,the time consumption,temperature,pH value of the solution and alcohol concentration during the extraction were optimized. The crude products were deprived of protein,pigment and ion,then were purified with DEAE-cellulose ion-exchange chromatography and verified with Sephadex G-100 and cellulose acetate membrane electrophoresis,and examined with infrared spectrum.

RESULTSThe optimized extraction conditions were as follows: extraction time 6 h,extraction temperature 70 degree,the solution pH 8.0 and the concentration of alcohol precipitation 70%. Intuitive features showed that the MIAP was pure white crystalline granular with slight dark brown color. The purification results demonstrated that the target MIAP was eluted and identified as a homogeneous components by DEAE-cellulose ion exchange column,Sephadex G-100 and cellulose acetate membrane electrophoresis. Infrared spectral scanning suggested that MIAP was α-D-type terminated glucopyranose. Intuitive features showed that MIAP was soft and cottony white.

CONCLUSIONThe extraction process with orthogonal test has been optimized and the acidic polysaccharide from Moerella iridescens is successfully isolated.

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45degrees C and the highest activity was exhibited in 35 to 45degrees C. The enzyme lost their activities almost completely (95~100%) at 80 degrees C or above and as well as bellow 25degrees C.
Acrylamide ; Aspergillus ; Aspergillus oryzae ; Benzyl Alcohols ; Cellulase ; Cellulose ; Chromatography, DEAE-Cellulose ; Chromatography, Gel ; Chromatography, Ion Exchange ; DEAE-Cellulose ; Electrophoresis, Polyacrylamide Gel ; Glucosides ; Hydrogen-Ion Concentration ; Molecular Weight ; Oryza

Inositol 1,4,5-triphosphate(InsP,) is a second messenger for obilizing intracellular Cal'. It can be dephosphorylated by soluble and particulate forms on InsP, 5-phosphatase, or phosphorylated to produce inositol 1,3,4,5-tetrakisphosphate(InsP,) by InsP, 3-kinase. These enzymes represent possible targets for the regulation of the InsP,AnsP. signal. InsP, 3-kinase which catalyses th ATP-dependent phosphorylation of InsP, was purified from bovine brain tissue. All operation were carried out at 41C. Fresh tissure was homogenized and centrifuged. The supernatant was pooled. Proteins were precipitated from 10% polyethylene glycol, and suspended solution was applied to DEAE cellulose column for chromatography. As the result of above procedure, two isozymes of InsP, 3-kinase, I and U were obtained. Each isozyme was applied to Matriz green gel, Calmodulin-Affigel 15 column and subsequent phenyl-TSK HPLC column. Specific activites(SA) and fold of puriety were observed at each purification step of chromatography. At DEAE cellulose chromatography, SA were 1, 0.6 and 11, 4.8 nM/min/mg, and folds
Brain* ; Catalysis ; Chromatography ; Chromatography, DEAE-Cellulose ; Chromatography, High Pressure Liquid ; DEAE-Cellulose ; Inositol* ; Isoenzymes ; Phosphorylation ; Phosphotransferases* ; Polyethylene Glycols ; Second Messenger Systems

1. Using the kidney of the rabbit weighing approximately 1.5kg the LDH isozymes, LDH-l through 5, were analyzed on the acetate strip by electrophoresis and it was found that the LDH-3 was the least active isozyme followed by the LDH-2, -4, -1, and -5 in increasing order of activity both in that cortex and the medulla, showing a specific distribution pattern in the two tissues. 2. The difference in the isozymic distribution pattern between the cortex and the medulla was confirmed by separating the H-and M-LDH by DEAE-cellulose, the ratio of M-/H-LDH being l. 441t the medulla and 3. O3 in the cortex, showing more active anaerobic metabolism in the latter than in the former. 3. The total activity of LDH in the 20% homogenate of the total kidney tissue was 10. 55 units/ml the cortical 2.98 units/ml. and the medullary 7. 37 unite/ml. 4. In the unilaterally nephrectomized rabbit. the cortex of the remaining kidney showed increased activities of both H- and M-LDH, but the medullary H-LDH decreased, resulting in increases of the ratio M-,H-LDH both in the cortex and in the medulla 5. In the unilaterally pedicle-clamped rabbit, LDH of the clamped kidney medulla decreased while that of the cortex increased, and LDH of the unclamped kidney showed elevated activity both in the cortex and in the medulla, yielding an increase in the ratio of M-/H-LDH. 6. The specific difference in distribution pattern of LDH igozymes between the cortex and the medulla of the rabbit kidney and an increase of the ratio of M-/H.LDH resulting from functional compensation following unilateral nephrectomy or pedicle clamping have been briefly discussed.
Compensation and Redress ; Constriction ; DEAE-Cellulose ; Electrophoresis ; Isoenzymes ; Kidney Medulla ; Kidney* ; Metabolism ; Nephrectomy ; Oxidoreductases*

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.
Amidohydrolases ; isolation & purification ; metabolism ; Arthrobacter ; enzymology ; Bacterial Proteins ; isolation & purification ; metabolism ; Chromatography, DEAE-Cellulose ; methods

OBJECTIVE: Activities of nucleases (acid DNase and neutral RNase) and RNase inhibitor known to be involved in carcinogenesis and suppression of cancer were determined in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma and were compared with those of the controls. Also studied were nucleases and RNase inhibitor isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients to evaluate the properties and interactions between them. METHOD: Activities of nucleases and RNase inhibitor were measured in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma by ultraviolet spectrophotometry. Nucleases and RNase inhibitor were isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients by DEAE-cellulose column chromatography. As controls, normal tissue of the cancer patients, serum of healthy persons and ascitic fluid of cirrhotic patients were used. RESULT: Activities of DNase, RNase and RNase inhibitor were significantly increased in hepatocellular carcinoma tissue. DNase activity was not detected, RNase activity was increased and RNase inhibitor activity was unchanged in both serum and ascitic fluid of the hepatocellular carcinoma patients. DNase was isolated as a single enzyme and RNase as seven isozymes from the hepatocellular carcinoma tissue. The DNase isolated preferentially cleaved ds DNA over ss DNA and was endonuclease in nature (majority of hydrolytic products of DNA by the DNase were oligodeoxyribonucleotides). Of seven RNase isozymes isolated from the hepatocellular carcinoma tissue, isozyme I exhibited nonsecretory nature of RNase and other six isozymes secretory nature of the enzyme. Activity of RNase isozyme V was greatly increased and the activity of inhibitor complexed with the isozyme V was also increased. RNase in ascitic fluid of the cancer patient was separated into four isozymes, of which isozyme I exhibited mixed form of secretory and nonseretory nature and greatly increased in its activity. RNase isozyme V isolated in the hepatocellular carcinoma tissue was not detected in the ascitic fluid. CONCLUSION: The use of the nucleases and the inhibitor in the cancer tissue as biochemical markers for the hepatocellular carcinoma was suggested. RNase was released into the body fluid from the cancer tissue and could be used as a diagnostic marker for the hepatocellular carcinoma. An important role of the DNase in carcinogenesis of the liver was suggested. RNase isozyme V was limited in the cancer tissue and RNase isozyme I and V and inhibitors associated with these isozymes might be involved in carcinogenesis processes, suppression of cancer and maintenance of hepatocellular carcinoma through their interactions.
Ascitic Fluid ; Biomarkers ; Body Fluids ; Carcinogenesis ; Carcinoma, Hepatocellular* ; Chromatography ; DEAE-Cellulose ; Deoxyribonucleases* ; DNA ; Humans ; Isoenzymes ; Liver ; Ribonuclease, Pancreatic ; Ribonucleases* ; Spectrophotometry, Ultraviolet

Cytochrome P-450 (CP-450) is one of the three components of the liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons and a variety of drugs and other foreign compounds. Female Balb/c mice were immunized with purified polychlorinated biphenyl (PCB) – induced CP-450 LMII. The spleen cells derived from immunized mice were fused with SP2 myeloma cells using polyethylene glycol (PEG 3500). The hybrid cells were selected by hypoxanthine-aminopterin-thymidine (HAT) medium and the culture fluid were screened by enzyme-linked immunosorbent assay to CP450 LMII. The hybrid cells(×107) were inoculated into intraperitoneal cavity of Balb/c mice for the purpose of production of ascetic fluids. Monoclonal antibody (Mab) was purified from ascitic fluid by DEAE cellulose ion exchange chromatography and I¹²⁵ labeled Mab was also confirmed by autoradiography and SDS-polyacrylamide gel electrophoresis (MW:55,000 and 110,000)
Animals ; Ascitic Fluid ; Autoradiography ; Chromatography, Ion Exchange ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; DEAE-Cellulose ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; Fatty Acids ; Female ; Humans ; Hybrid Cells ; Hydrocarbons ; Liver* ; Mice ; Polyethylene Glycols ; Rats* ; Spleen

OBJECTIVE: The continuous synthesis and degradation of proteins in the cell are essential for the maintenance of cellular homeostasis. Intracellular protein degradation largely occurs in the lysosome and cytoplasm. The protein degradation in the cytoplasm (ubiquitin mediated protein degradation) is distinct from the well studied lysosomal protein degradation (nonselective protein degradation) and require energy (ATP), ubiquitin and ubiquitin conjugating enzymes such as E1, E2 and E3. Dementia caused by the deposition of abnormal proteins in brain cells followed by brain cells damage are not fully understood. To better understand the possible mechanism of dementia, we attempted to purify ubiquitin conjugating enzymes (such as E1 and E2 proteins) from the blood of normal persons and patients with dementia and tested their electrophoretic mob)ility on SDS-polyacrylamide gel electrophoresis. METHOD: The E1 and E2 enzymes of the red blood cell lysate fraction from the normal person and the patients with dementia were purified from ammonium sulfate precipitatant of DEAE-cellulose eluate fraction. Following ubiquitin-sepharose column chromatography, the E1 enzyme of the normal and the patients with dementia group showed homogeneous form and various kinds of E2 isoforms were identified by the SDS-polyacrylamide gel electrophoresis. RESULTS: The E1 and E2 enzymes showed no difference on electrophoretic mobility, but the E2 isozyme containing fraction was observed to great difference between the two groups. The 44 kDa protein of E2 isozyme containing fraction was significantly increased in alcoholic dementia and clearly increased in patients with Alzheimer's disease. In addition, another 11 kDa protein was significantly increased in the patients with Alzheimer's disease, but 11 kDa protein of alcoholic dementia was similar to that of the normal person. The 44 kDa and 11 kDa proteins showed a reverse relationship between alcoholic dementia and the patients with Alzheimer's disease. These proteins seems to be different molecules from the well known studied beta-amyloid, presenilin, tau protein and apolipoprotein E (Apo E). CONCLUSIONS: These results might be useful for the elucidation of dementia and the identification of these proteins are now in progress.
Alcoholics ; Alzheimer Disease ; Ammonium Sulfate ; Apolipoproteins ; Brain ; Carrier Proteins* ; Chromatography ; Cytoplasm ; DEAE-Cellulose ; Dementia* ; Electrophoresis ; Erythrocytes* ; Homeostasis ; Humans ; Lysosomes ; Presenilins ; Protein Isoforms ; Proteolysis ; tau Proteins ; Ubiquitin* ; Ubiquitin-Conjugating Enzymes

Guanine aminohydrolase(GAH;EC 3. 5. 4. 3.) was partially purified 122-fold from rat cerebrum to a specific activity of 7.22 in its per mg protein with a recovery of 7.47% by fractionation with ammonium sulfate, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and isoelectric focusing(pH4-6). The molecular weight of partially purified rat cerebral guanine aminohydrolase was estimated to be 110,000. But, in the cerebral cytosol, a rather higher molecular weight form of the enzyme was identified. The activity of the higher molecular weight form of guanine aminohydrolase was increased by dialyzing the cytosol, and it was converted into the lower molecular weight form(M.W.110,000) by addition of 2-mercaptoethanol. The reaction velocity of partially purified guanine aminohydrolase of rat cerebrum disclosed a hyperbolic curve, with its KM being 6.0uM at pH 8.0. The preparation showed high substrate specificity:among the purine nucleotides, nucleosides and bases with amino group, only guanosine and guanine were deaminated by the enzyme, and the reaction rate of the enzyme displayed by guanosine was less than 10% of that by guanine. When observed under the equimolar concentration of the substrate, hypoxanthine as well as inosine inhibited the activity of the rat cerebral guanine aminohydrolase by 9.4 and 7.8%, respectively, while 5-aminoimidazole-4-carboxamide inhibited the activity of it by 38%. The activity was inhibited by p-hydroxymercuric benzoate as well. Complete loss of its activity was observed after 30 minutes incubation at 60 degrees C, suggesting the preparation was heat labile.
Ammonium Sulfate ; Animals ; Benzoates ; Cerebrum* ; Chromatography ; Chromatography, Gel ; Cytosol ; DEAE-Cellulose ; Durapatite ; Filtration ; Guanine Deaminase* ; Guanine* ; Guanosine ; Hot Temperature ; Hydrogen-Ion Concentration ; Hypoxanthine ; Inosine ; Mercaptoethanol ; Molecular Weight ; Nucleosides ; Purine Nucleotides ; Rats* ; Xanthine Oxidase