Ganglioside sialidase distribution in mucolipidosis type IV cultured fibroblasts

The subcellular distribution of ganglioside sialidase in Mucolipidosis IV (ML IV) cells was characterized by a series of Percoll gradients. Similar to normal cells, the enzyme cosedimented with plasma membrane markers, although this activity was reduced and exhibited decreased solubility in ML IV cells. Only trace amounts of ganglioside sialidase (less than 5%) was found in the lysosomes of normal cells. This activity was apparently reduced in ML IV cells but its minute activity in controls excluded further characterization of these differences. Plasma membranes on 6.7 and 5.6% Percoll gradients were biomodally distributed. Ganglioside sialidase in normal cells was found to be in both the heavier and the lighter membrane fractions, whereas the enzyme in ML IV cells was associated mainly with the denser membrane fraction. These data indicate that the enzyme in ML IV cells is characteristically different from normal in that it exhibits reduced activity and solubility and a different plasma membrane distribution.     

A case of 21q-syndrome with half normal SOD-1 activity

A male Japanese infant was found to have a chromosomal aberration of del(21)(qter leads to q22.1-2) and decreased superoxide dismutase (SOD) activity in erythrocytes and polymorphonuclear and mononuclear leukocytes. The cuprozinc enzyme (SOD-1) level was 40-50% of normal, while the cyanide-insensitive manganese enzyme (SOD-2) activity was within the normal range. Determination of SOD activity in blood cells is a valuable method of classification of the syndrome.     

Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease

Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.     

Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

An inhibitor of host sigma-stimulated core enzyme activity that purifies with DNA-dependent RNA polymerase of E. coli following T4 phage infection

The content of the sigma subunit (as detected by gel electrophoresis) and activity with T4 DNA were examined with RNA polymerase fractions from both normal and T4 phage-infected $\text{E. coli}$. Sigma-containing fractions and core enzymes were obtained by phosphocellulose column chromatography. The sigma-containing fraction of the enzyme from infected cells, although somewhat stimulatory to both core enzymes alone, inhibits the normal sigma-stimulated activity of the core enzyme from infected cells at both low and high KCl concentration. Normal core enzyme activity is inhibited only at high KCl concentration.     

Properties of a murine monocytic tumor cell line J-774 in vitro. II. Enzyme activities.

Lysosomal enzyme activities, collagen degrading activity and sensitivity to bacterial infection were tested in a murine monocytic cell line, J-774, during cultivation with or without fetal calf serum (FCS) or endotoxin, and compared with the same parameters in normal murine peritoneal macrophages. The basic intracellular level of two out of three lysosomal enzyme activities tested (acid phosphatase and β-glucuronidase) and their extracellular release were higher in the J-774 cells than in normal macrophages, indicating that the tumor cells were more “activated”. This was further supported by the moderate increase in intracellular enzyme activities after FCS and endotoxin stimulation of the J-774 cells. Normal macrophages showed a much more impressive rise in these parameters after stimulation. Collagen-degrading activity was found at the same magnitude, or lower, in tumor cell cultures, compared to normal macrophage cultures. However, the activity in the tumor cultures was enhanced by endotoxin stimulation. The J-774 cells showed a higher sensitivity to bacterial contamination, tested after E. coli addition to the cultures, than normal macrophages. This high sensitivity could be prevented by pretreatment of the tumor cells with endotoxin.     

Temporal order in mammalian cells. I. The periodic synthesis of lactate dehydrogenase in the cell cycle

Chinese hamster cells were synchronized by the Colcemid-selection system. In cells with a division cycle time of 11.5–12 hr, the activity of the enzyme lactate dehydrogenase (LDH) underwent marked oscillations with a 3.5-hr period. Precipitation of labeled LDH enzyme with specific antibody indicated that the enzyme activity changes were the result of intermittent enzyme synthesis and relatively constant degradation. Inhibition of normal DNA replication with 4 mM of thymidine, while reducing the amount of new enzyme synthesized, did not prevent oscillations from occurring. Similarly, actinomycin D (AcD) added at the time of synchronization allowed some new enzyme synthesis to proceed in an oscillatory manner. LDH synthesis went on at nearly normal rates when AcD was added in the middle of S phase. However, addition of cycloheximide to cultures at any time in the cycle caused an immediate drop in levels of activity and in enzyme protein. The half-life of LDH, calculated either from loss of enzyme activity or precipitable radioactivity in cycloheximide-treated cultures, was between 2 and 2.5 hr.     

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity

The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.     

根瘤菌BK-20固定化细胞生产L(+)-酒石酸

顺式环氧琥珀酸水解酶(CESH)是根瘤菌BK-20生产L(+)-酒石酸的关键酶。为提高其生产效率和生产稳定性,首先优化根瘤菌BK-20的产酶条件,然后利用固定化细胞连续生产L(+)-酒石酸。结果显示,优化后游离细胞酶活达(3 498.0±142.6)U/g,较优化前提高643%。固定化细胞酶活达(2 817.2±226.7)U/g,其最适包埋剂、菌体浓度和凝胶浓度分别为海藻酸钠,10%(W/V)和1.5%(W/V)。固定化细胞连续反应10批后,其形状和酶活均无明显改变,单批次转化率达98%以上,具有良好的生产稳定性。     

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K_m ATP or a change in the kinetics of Mg⁺⁺ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.     

N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt

N-Acetyl-glucosaminidase activity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12–15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.     

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.     

Improved hepatocyte culture system for studying the regulation of glycogen synthase and the effects of diabetes

When 3–4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (>150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitaion of ³⁵S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes. © 1996 Wiley-Liss, Inc.     

Acid alpha-D-mannosidase of human leukocytes in the norm and during chronic myeloid leukemia

The activity and properties of acid alpha-mannosidase were studied in normal granulocytes and in two types of myeloid cells from patients with chronic myeloid leukemia. The activity of the enzyme in leukemic cells was 2-fold higher than that in normal granulocytes and in morphologically matured myeloid cells. Two latter types of cells did not differ in alpha-mannosidase activity. Kinetic properties, thermo- and pH stability of alpha-mannosidase from normal and leukemic cells were similar. alpha-mannosidase in leukemic and normal cells existed in two forms (A and B), which were easily separated on DEAE-cellulose column. These two forms differed in molecular mass (300 and 290 kD, respectively) and in the degree of sialylation. The quantitative ratios of A and B forms in normal and leukemic cells were different. In normal granulocytes and in mature cells from patients this ratio was 0.60 and 0.67, respectively. In leukemic cells the ratio was found to be 1.31. Thus, in leukemic cells form A of alpha-mannosidase predominanted, whereas in normal cells the predominance of form B was observed. It was suggested therefore that in leukemic cells the enhanced synthesis of alpha-mannosidase occurred in parallel with the accumulation of the B form. This accumulation was assumed as the cause of enhanced activity of the enzyme in immature leukemic cells.     

Alteration of beta-hexosaminidase activity and isoenzymes in human leukemic cells

beta-Hexosaminidase (EC 3.2.1.20; Hex) activity and isoenzyme characteristics were analyzed in human normal and leukemic leukocytes. Unseparated CLL and CML cells had a specific activity that was lower, whereas ALL and AML blasts had a higher specific activity than normal lymphocytes and granulocytes. CLL B-cells had a lower specific activity compared with that in normal non-T-lymphocytes; CLL T-cells and normal T-cells had similar activity. Isoenzyme separation was performed by chromatofocusing on PBE-94 coupled with an automated enzyme assay. When using a single linear pH elution gradient, normal leukocytes and all leukemia cells contained two forms of isoenzyme (B and A). When a double pH elution gradient was performed, an extra distinct form of Hex (I) was recorded. Hex I was present in small amounts in normal granulocytes and PHA-stimulated normal lymphocytes; isoenzyme I was found in high amounts in all leukemias tested. The activity ratios I/B and I/A, as well as the I isoenzyme profile, may facilitate differentiation between normal and leukemic cells and between lymphoblastic and myeloblastic leukemias.