Thermally perturbed rhodanese can be protected from inactivation by self-association

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30–40°C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 µg/ml is inactivated at 40°C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 µg/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30–40°C. Light scattering kinetics at 40°C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions: at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Gluteraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40°C but not at 23°;C. Precipitates formed at 40°C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.     

Investigation on supraphysiological thermal injury in two well-differentiated human hepatoma cell lines,HepG2 and Hep3B

Hyperthermia is a promising treatment for carcinoma cells. The thermal injuries of two hepatoma carcinoma cell lines with the identical cytological grade, HepG2 and Hep3B cell lines, were investigated systematically in the present study. The homemade heating stage was used to provide a constant temperature between 40 and 70 °C for thermal treatment. When the cells were exposed to temperatures ranging from 40 to 45 °C, Hep3B cells had a lower thermotolerance than the HepG2 cells; however, the survival rate of these two cell lines was still high. The differences in thermotolerance between HepG2 and Hep3B cells were more significant at the range of 50–55 °C than those at lower-level temperatures of 40–45 °C. Furthermore, the viability of the cells was less than 10% when they were exposed to a supraphysiological temperature of 60 °C for 5 min; these cell lines suffered from injury saturation under that thermal treatment. The statistical analysis also concluded that Hep3B cells are more susceptible to heat stress than are the HepG2 cells when subjected to the thermal treatment applied in this work, the exception being when thermal injury saturation occurred. The kinematic parameters of the activation energy and frequency factor for HepG2 and Hep3B cells were also quantitatively determined herein. The activation energies (ΔE) for HepG2 and Hep3B cells were 170.17 and 152.44 kJ/mol, respectively. Furthermore, the frequency factors (A) for HepG2 and Hep3B cells were 4.11×10²⁴ and 1.07×10²² s⁻¹, respectively.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.     

On the size and chemical nature of the polypeptide chain of bovine liver rhodanese.

Evidence from molecular weight studies and sequence analysis of bovine liver rhodanese indicates that the enzyme is a single polypeptide of molecular weight 35,200, and not a dimer of identical subunits half this size. The rhodanese molecule contains 317 amino acids including 5 methionines, 4 cysteines, and 5 tryptophans. As expected, six fragments were produced by cleavage with cyanogen bromide and these have been aligned in the enzyme with the aid of overlapping tryptic peptides isolated from a [¹⁴C] carboxymethylmethionyl rhodanese derivative. The cyanogen bromide fragments account for all of the amino acid residues of the parent rhodanese molecule. Methionine residues are located at positions 72, 112, 214, 217, and 235 in the polypeptide chain and the active site cysteine is at position 251, in the carboxyl-terminal segment of the molecule.     

STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury

Interferon-gamma (IFN-gamma) has been implicated in liver damage in animal models and chronic hepatitis C infection; however, the underlying mechanism is not clear. Here we examined the role of STAT1, a key signaling molecule for IFN-gamma, in a model of murine hepatitis induced by the injection of LPS/D-galactosamine and in human hepatoma Hep3B cells. STAT1 is rapidly activated and highly induced after injection of LPS/D-galactosamine. Both overexpression of STAT1 and hepatocellular damage are located in the same pericentral region. Disruption of the STAT1 gene abolishes LPS/D-galactosamine-induced liver injury. Studies from IFN-gamma-deficient mice indicate that IFN-gamma is the major cytokine responsible for activation and hyperexpression of STAT1 in LPS/D-galactosamine-induced hepatitis. Hep3B cells overexpressing dominant negative STAT1 are resistant to IFN-gamma and IFN-gamma + TNF-alpha-induced cell death, whereas Hep3B cells overexpressing wild-type STAT1 are more susceptible to cell death. Taken together, these findings suggest that STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury.     

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase")

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.     

Studies on rhodanese synthesis in bovine adrenocortical cells

The synthesis of adrenodoxin, a mitochondrial iron-sulfur protein required for adrenocortical steroidogenesis, is known to be regulated chronically by ACTH. Rhodanese, also a mitochondrial enzyme, is thought to be required for synthesis of iron-sulfur centers, such as those contained in adrenodoxin. In this study it has been found that rhodanese synthesis and activity are not regulated by ACTH, under the same conditions whereby ACTH induces adrenodoxin synthesis. In addition, unlike adrenodoxin, rhodanese is found to be synthesized in the mature form rather than as a higher molecular weight precursor protein.     

Evidence for conversion of human salivary alpha-amylase family A to family B by an enzyme action.

SDS-PAGE showed that human salivary alpha-amylase family A (HSA-A) was converted to family B (HSA-B) in human saliva. This conversion did not occur in the supernatant of saliva which had been centrifuged at 105,000 x g for 60 min. An enzyme which catalyzed the conversion existed in the insoluble fraction of human saliva. The enzyme was solubilized with nonionic or zwitterionic detergents, and showed the maximum activity around pH 6. It was stable between pH 4 and 10, and at a temperature lower than 40 degrees C. The enzyme reduced the molecular weight of HSA-A (62,000) to the same molecular weight (58,000) as that of HSA-B without forming any intermediate. It also changed the PAGE pattern of multiple forms of HSA-A to the same pattern as that of HSA-B. It was not inhibited by protease inhibitors, and it did not destroy the reactivity of HSA-A with anti-human salivary alpha-amylase antiserum. The enzyme diminished the reactivity of HSA-A with concanavalin A. These results indicate that HSA-A was converted to HSA-B through the release of sugar chains by the action of the enzyme in the insoluble fraction of human saliva.     

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.     

Multivariate factor analysis of sulfur oxidation and rhodanese activity in soils

The role of rhodanese as an intermediate catalyst in the oxidation of elemental S (S°) is not well understood. This study investigated the effect of 26 soil properties and steam sterilization in relation to S° oxidation and rhodanese activity in 33 soils (27 Oregon soils and six Chinese soils). S° oxidation potential was determined by incubating (7 d at 23 °C) soil amended with 500 mg S° kg^-1 soil and measuring the SO₄ released. Both total S° oxidation (TSO) and rhodanese activity varied widely among the 33 soils, ranging from 0 to 143 mg SO₄-S kg^-1 soil 7 d^-1 and 22 to 2109 nmoles SCN^- g^-1 soil h^-1 respectively. S° oxidation but not rhodanese activity had a significant positive correlation with soil pH. In sterile soils, chemical S° oxidation (CSO) averaged 3% of the total S° oxidation and apparent rhodanese activity averaged 11% of the total rhodanese activity. S° oxidation was not significantly correlated with rhodanese activity. However, development of stepwise regression models predicting S° oxidation revealed that rhodanese activity was an important explanatory variable in predicting biological S° oxidation (TSO minus CSO). Also, microbial biomass C was found to be an important parameter in models for both S° oxidation and rhodanese activity. Investigations of the effect of acidification during S° oxidation showed that biological S° oxidation was negatively correlated with S° oxidation-induced-pH-change for soils with pH > 6 but no such significant relationship was found on soils with pH> 6. This suggested that extreme acidity may inhibit S° oxidation but not rhodanese activity.     

Accumulation of peptides by mycobacillin-negative mutants of Bacillus subtilis B3

Thirteen mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. The wild-type producer, three feeble producers and three strictly My- mutants did not accumulate any ninhydrin-positive peptide in the culture medium while the remaining seven My- mutants did accumulate ten such peptides whose amino acid composition indicated that there might be only three different peptides. The N-terminal and C-terminal amino acid residues implicated one of these peptides as a pentapeptide intermediate in mycobacillin synthesis; this was further confirmed by its molecular weight and sequence. Studies on cell-free synthesis showed that only the enzyme system from the wild-type strain synthesized mycobacillin while the defective ones from all the My- mutants synthesized one and the same pentapeptide as found in the culture broth of some of the mutants. Further studies in which the enzymes responsible for mycobacillin synthesis by cell-free extracts were separated into three fractions, A, B and C, showed that seven of the mutants were defective in fraction B whereas the three other mutants had defects in both fractions B and C. Thus the pentapeptide Pro----Asp----Glu----Tyr----Asp appears to be implicated in mycobacillin biosynthesis.     

Expression of cloned bovine adrenal rhodanese

A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.     

Characterization of UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B.

UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1, 6-N-acetylglucosaminyltransferase V (GlcNAcT-V) has been purified from cell extracts of the human hepatoma cell line, Hep3B, with 8.7% recovery. The purified enzymes had molecular masses of about 67 and 65 kDa on denaturated and natural conditions, respectively. The values of pI was 5.9. The GlcNAcT-V, when resolved by SDS-PAGE, was positive for Schiff staining, suggesting that the enzyme is glycoprotein. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzyme displayed a temperature optimum of around 50 degrees C and optimum an pH of 6.5. The enzyme was stable in response to incubation from pH 4.5 to pH 10.5 at 4 degrees C for 24 h. The presence of UDP-GlcNAc and GlcN,GlcN-bi-PA protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ion; however, it was inhibited by Fe3+. The enzyme activity was inhibited by another series of NDP-sugars including ADP-, CDP-, GDP-, and TDP-GlcNAc. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward biantennary (GlcN,GlcN-bi-PA) sugars. The enzymes had apparent Km values of 1.28 and 5.8 mM for GlcN,GlcN-bi-PA and UDP-GlcNAc, respectively. In order to isolate the GlcNAcT-V gene, PCR primers of GNN-1 and GNN-8 were designed and the amplified PCR product carrying the gene was cloned and sequenced. Nucleotide sequence analysis showed a 2220-bp open reading frame encoding a 740-amino-acid protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. The three amino acid changes were as follows: 375V --> L, 555T --> R, and 592A --> G. These studies represent the detailed characterization of a purified GlcNAcT-V from human hepatoma cell Hep3B.     

A study of the single polypeptide nature of rhodanese. A comparison of different preparations.

The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Rhodanese activity and total sulfur content in frog and mouse liver

The activity of rhodanese was histochemically tested in cryostat sections of the frog (Rana temporaria) and mouse liver. The activity levels were evaluated in sections, and the results were expressed as the ratio of the area of all granules, products of the enzymatic test, to the total analyzed area (area fraction). The present study confirmed the biochemically detected activity of rhodanese, and showed a large pool of endogenous sulfane sulfur donors, substrates for rhodanese, in the frog liver. The area of a single granule corresponded to the size of the mitochondrium, what suggests enzyme localization in this organelle. In view of this it should be considered whether the rhodanese activity, biochemically detected in the cytosolic fraction of the frog liver, results from the enzyme action. The total content of sulfur in cryostat sections of the mouse and frog liver was calculated and compared on the basis of the Energy Dispersion Spectrum (EDS) obtained by a scanning microscope. These studies showed higher total sulfur content in the frog liver than in the mouse liver. The high total content of sulfur in the frog liver in autumn might be associated with sulfur storing for protein biosynthesis during the period of hibernation.     

Cobalt-activated aminopeptidase fromStreptococcus sanguis NCTC10904

An aminopeptidase fromStreptococcus sanguis NCTC 10904 was isolated, purified, and characterized. The enzyme was produced constitutively and could be isolated from the cytoplasmic fraction of lysed cells. Its substrate profile indicated that it is primarily a leucyl aminopeptidase, but with a substrate spectrum including lysyl- and arginyl-peptides. The subunit molecular weight of the enzyme was approximately 74,000, but an octomeric form also was prominent, as indicated by gel filtration separations of active enzymes. The optimal temperature for activity was 32°C, and the optimal pH value was about 7.0. The enzyme showed cooperative kinetics and was activated by Co²⁺. The regulation of synthesis and the characteristics of the enzyme suggest that it may serve a regulatory function rather than just a nutritional function.