Heat Inactivation Studies Research Articles

Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk − and LMtk − cells with 5-iodo-5′-amino-2′,5′-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5′-amino-5′-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2′-deoxyuridine (IdUrd) caused a similar increase only in tk − cells (LMtk −, BHKtk −). In vivo and in vitro stabilization studies using a [ 35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects.

Polyphenoloxidase from guava (Psidium guajava L.)

AbstractPolyphenoloxidase (PPO) was isolated from guava (Psidium guajava L., cv. Kampuchea). Purification by acetone precipitation and dialysis of the Amicon concentrate resulted in 4.9‐fold and 17.2‐fold purification of the dialysed crude extract respectively. Polyacrylamide gel electrophoresis indicated that PPO had a molecular weight in the region of 120000–140000. The pH optimum of the enzyme was found to be 7.2. Heat inactivation studies showed that heating for 1.1, 3.5, and 10.1 min at 75, 65 and 55°C respectively caused a 50% loss in enzymic activity. The enzyme‐catalysed browning reaction was significantly inhibited in the presence of sodium metabisulphite, L‐cysteine hydrochloride and ascorbic acid.

Glycerol Oxidation and Triose Reduction by Pyridine Nucleotide-linked Enzymes in the Fission Yeast Schizosaccharomyces pombe

Summary: The fission yeast Schizosaccharomyces pombe has been shown to produce four separate pyridine nucleotide-linked glycerol dehydrogenases (or triose reductases), distinguished by differences in their coenzyme specificity (NAD+ or NADP+) and oxidation product (dihydroxyacetone or glyceraldehyde). Evidence for four separate activities was obtained by heat inactivation studies, comparison of cells grown under different conditions, and separation and partial purification of the enzymes. One enzyme, a glycerol: NAD+ 2-oxidoreductase is repressed by glucose but not induced by glycerol and appears to function primarily in glycerol catabolism. The second, a glycerol: NADP+ 2-oxidoreductase is stimulated by growth on glucose and appears to function as a dihydroxyacetone reductase involved in glycerol synthesis. The third has the properties of a glycerol: NADPT+ oxidoreductase, while the fourth is, in fact, alcohol dehydrogenase (alcohol: NAD+ oxidoreductase) which possesses weak activity as a glycerol: NAD+ 1-oxidoreductase.

Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein

The interaction of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein, infected-cell polypeptide 8 (ICP8), with nucleic acids has been examined by a filter-binding assay and electron microscopy. Filter-binding assays done over a broad pH range indicated that the optimum pH for the protein-DNA interaction is approximately 7.6. Heat inactivation studies showed that ICP8 is stable at temperatures up to 40 degrees C with a rapid loss of binding activity on incubation at 45 degrees C and above. Competition binding experiments have established the following relative affinities of ICP8 for the following nucleic acids: single-stranded HSV-1 DNA congruent to bacteriophage fd DNA greater than polyriboadenylate much greater than double-stranded HSV-1 DNA congruent to d(pCpT)5. Observation of negatively stained ICP8-single-stranded DNA complexes indicated that ICP8 binds along the length of the DNA in a regular repeating fashion. The average width of these complexes is 9.3 +/- 0.8 nms. Finally. Finally, addition of purified ICP8 to HSV-1 DNA polymerase reactions resulted in a stimulation of the viral polymerase activity.

The effects of in vitro ageing on the exopeptidases of human diploid fibroblasts

Six exopeptidases present in human diploid fibroblasts were identified by separation on polyacrylamide gel electrophoresis and their activity profiles against 17 dipeptides, two tripeptides and l-leucine -p- nitroanilide determined. No differences in relative activity or in the electrophoretic patterns of any of the six exopeptidases were detected with ageing. Aminoacylarylamidase activity assayed spectrophotometrically showed significantly increased activity in the middle age-group cells as opposed to the enzyme isolated from young and old cells. Heat-inactivation studies using the same substrate suggested the possibility of an increased proportion of heat-labile enzyme in the old cells but interpretation of the data was difficult because of the complex nature of the inactivation curves obtained. Overall, the results tended to refute the hypothesis that age-related changes in the free amino acid pool of human diploid fibroblasts were associated with significant alterations in the activities of cellular exopeptidases.

Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease.

An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.

In-vitro transcription in tobacco chromatin

A method is described to isolate transcriptionally active Nicotiana tabacum L. cv. Xanthi chromatin from suspension-cultured cells. Enzymatic preparation of protoplasts with solubilization of the plasma membrane, Triton X-100 and homogenization resulted in chromatin free from cellular debris. Incroporation of [(3)H]uridine triphosphate into RNA increased for more than 30 min at 30° C. Transcriptional activity was maximally stimulated at 10 mM MgCl2, 200 mM (NH4)2SO4 and 150 mM KCl. The in-vitro synthesized RNA was found to contain 3.8% polyadenylated RNA. The results of digestion studies with ribonuclease, heat and detergent inactivation studies, α-amanitin and actinomycin D inhibitor effects, as well as dependency on ribonucleotide triphosphates, showed that the activity recorded in the tobacco chromatin was bona-fide enzymatic RNA transcription. There also was marked stimulation of transcriptional activity when exogenous DNA was added to the assay mixture.

Two macromolecular complexes between alkaline phosphatase and immunoglobulin A in a patient's serum.

We report the presence of two separate macromolecular complexes between an immunoglobulin and alkaline phosphatase (EC 3.1.3.1) in the serum of a patient with cholestatic liver disease. The complexes were seen as slow-moving bands on electrophoresis on polyacrylamide gel (70 g/L). Estimates of relative molecular mass for the two bands were 785 000 and 490 000 by gel chromatography on Sephacryl S300, and 640 000 and 420 000 by gradient gel-electrophoresis. Inhibitor and heat-inactivation studies showed that the two bands had similar properties, resembling the liver isoenzyme but more heat stable. Immunological studies suggested that both bands consisted of a complex of liver alkaline phosphatase with kappa type immunoglobulin A.

Partial Purification and Characterization of Cystine Lyase from Cabbage (Brassica oleracea var capitata)

Cabbage (Brassica oleracea var capitata) leaves were used as a source of cystine lyase. Diethylaminoethyl-cellulose chromatography resolved two peaks of activity, designated I and II.Cystine lyase I (molecular weight 145,000) and O-acetylserine sulfhydrylase (molecular weight 70,000) were resolved by Bio-Gel A-0.5M chromatography. This isozyme catalyzed an alpha,beta-elimination reaction with cystine, cysteine, O-acetylserine, and several S-substituted cysteines. The substrate specificity was similar to previously reported S-alkylcysteine lyases. The elution profiles during purification, and heat inactivation studies indicated that the above reactions were catalyzed by a single protein. The pH optimun with cystine and cysteine as substrate was 8.5 to 9.0, and the K(m) values were: cystine (0.3 mm), cysteine (0.3 mm), O-acetylserine (6 mm), and S-methylcysteine sulfoxide (1.8 mm).Cystine lyase II was resolved into three peaks (molecular weight greater than 500,000, 240,000, and 145,000) using Bio-Gel A-0.5M chromatography. This enzyme degraded l-cystine, l-cysteine, O-acetylserine, S-methylcysteine sulfoxide, and djenkolic acid. The pH optimum with cystine and cysteine was 8.5 to 9.0, and the K(m) values were: cystine (0.3 mm), cysteine (0.3 mm), O-acetylserine (12.5 mm), and S-methylcysteine sulfoxide (3.7 mm).

Mitochoudrial phenylalanylt-RNA synthetasc from yeast: Formation of enzyme-substrate complexes shown by heat or SH reagent inactivation

The binding of substrates to purified mitochondrial phenylalanyl-tRNA synthetase from yeast was examined using the kinetics of heat or p-hydroxymercurybenzoate inactivation. Individually magnesium chloride and each of the substrates protect the enzyme against thermal denaturation and p-hydroxymercurybenzoate inhibition. No enzyme protection is observed with ATP alone against p-hydroxymercurybenzoate inhibition. The combinations of the various substrates induce a synergistic protection effect. Protection constants of 31 microM and 0.3 microM were found for L-Phe and mt tRNAPhe respectively, from heat inactivation studies. The inhibition of the enzyme activity by p-hydroxymercurybenzoate can be reverted by 2-mercaptoethanol or dithiothreitol.

Isolation and Characterization of Lipoxygenase in Pinto Dry Beans

Pinto dry beans are often rejected for human consumption due to off flavours. A potential explanation of off flavour development could be the activation of lipoxygenase. In this investigation, Pinto dry bean lipoxygenase was isolated and characterized. The enzyme was found to have a pH optimum of 6.9 and a Km of 0.0556mM based on linoleic acid as the substrate. The production of hexanal, an indicator of lipid breakdown, was evaluated as a function of equilibrium relative humidity and was found to correlate with increasing moisture levels. Heat in-activation studies of the enzyme were conducted in aqueous solutions and lipoxygenase could be destroyed in 15 s at 100°C. Hexanal production in the beans was considered a strong indicator that off flavours in Pinto dry beans could be due to lipoxygenase activity during storage at higher than normal moisture contents.

Metal ions and clostridiopeptidase A

Clostridiopeptidase A is a collagenolytic enzyme produced by the bacterium Clostridium histolyticum. It contains four identical sub-units, each with a molecular weight of about 25,000. The enzyme contains Zn and requires Ca 2+ for activity [1]. We are using lanthanide ions (Ln 3+) and Co 2+ to investigate the role of Ca 2+ and Zn, respectively, in the catalytic activity of clostridiopeptidase A. For this purpose, it is convenient to use an artificial pentapeptide (‘Pz-peptide’) as the substrate in place of collagen whose properties are altered in the presence of Ln 3+ [2]. Previous kinetic analyses, conducted in this laboratory, of the effect of Ln 3+ on the hydrolysis of Pz-peptide by clostridiopeptidase A have suggested that Ca 2+ is required for the binding of enzyme and substrate [3]. Sm 3+ lowers the K m nearly 15 fold, but it also lowers the V max by similar amount, suggesting that the enzyme-substrate complex is an abortive one. The relative ability of different Ln 3+ to inhibit clostridiopeptidase A is Lu < Er < Sm ⪢ La. As the radius of La 3+ (1.016 Å) exceeds that of Ca 2+ (0.990 Å), while Sm 3+ (0.964 Å), Er 3+ (0.881 Å) and Lu 3+ (0.850 Å) are smaller, the Ca 2+-binding site on the enzyme appears to be sterically constrained, such that ions of greater radius than that of Ca 2+ have restricted access. Heat inactivation studies also revealed a thermostability role for Ca 2+ [3]. The ability of Ln 3+ to inhibit clostridiopeptidase A while enhancing its substrate-binding, suggests a role for these cations in the purification of this enzyme by affinity chromatography; previous attempts to do this have been foiled by collagenolysis, even at 4 °C [4]. Experiments to investigate this possibility have qualitatively confirmed the conclusions from the kinetic analyses mentioned in the preceding paragraph. When a Ca 2+-free solution of clostridiopeptidase A was passed through an affinity column packed with calf skin collagen trapped within particles of polyacrylamide [4], none of the enzyme stuck to the column. Addition of increasing amounts of Sm 3+ progressively enhanced binding, with complete sequestration of clostridiopeptidase A occuring at a Sm 3+ concentration of 75 μM. Comparison of the efficiencies of Lu 3+, Er 3+, Sm 3+ and La 3+ at a concentration of 75 μM (Table I), gave results in complete accord with those of the kinetic studies. Co 2+ enhanced the rate of hydrolysis of peptide by clostridiopeptidase A, a maximum stimulation of 2.5–3 fold being obtained with 2.5 mM Co 2+. Lineweaver-Burk analysis of this stimulation revealed an elevation of both the V max and K m by a factor of about 2.5 in the presence of 2.5 mM Co 2+. Taken at face value, these findings indicate that Co 2+ increased the catalytic efficiency of the enzyme, despite reducing its affinity for the substrate. If Co 2+ specifically replaced Zn, as happens with many other enzymes, the data suggest a previously unrecognized role for Zn in substrate-binding. However, we are also investigating possible alternative explanations, such as an action of Co 2+ at the Ca 2+-binding site of the enzyme, or its interaction with the substrate. Both Ln 3+ and Co 2+ provide useful spectroscopic properties which should greatly aid further investigation of these matters.

An approach to the elucidation of the quaternary structure role in the activity of pyruvate kinase studies on the immobilized enzyme

1. 1. Studies on pyruvate kinase immobilized via -S-S- linkages showed that single subunits of this tetrameric enzyme are inactive. 2. 2. Pyruvate kinase was coupled with different preparations of CNBr-activated Sepharose via one and more than one bond, and after removal of non-covalently bound subunits by denaturant treatment the subunit derivatives were obtained composed of different pyruvate kinase species. 3. 3. Heat inactivation studies of immobilized enzyme derivatives suggested that not only tetramers of pyruvate kinase are active. 4. 4. This idea is further supported by experiments with 2 M urea dissociation of immobilized tetrameric enzyme carried out in the presence of Mg 2+ ions.

Reverse transcriptase from simian foamy virus serotype 1: purification and characterization.

Chromatography on heparin-Sepharose, known for its affinity for nucleotide-binding polypeptides, was used to purify the viral RNA-dependent DNA polymerase (reverse transcriptase) from the core polypeptides of simian foamy virus type 1. This procedure allowed the recovery of highly purified enzyme with a high specific activity. The average molecular weight of this monomeric enzyme is 81,000 and is thus comparable to that found for other known primate retroviruses. Reverse transcriptase activity of simian foamy virus type 1 requires a ribonucleotide template as a primer or otherwise a DNA with 3'-OH ends. Other optimal conditions of activity are reviewed. Heat inactivation studies led to the concept of an enzyme with two loci, one specific for the substrate and the other for the template-primer.

Characterization of a new human cell line derived from a xenografted embryonal carcinoma.

A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressed beta 2-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.

Cyclic nucleotide phosphodiesterase activity in midpiece and tail of buffalo spermatozoa and its role in sperm motility

Cyclic nucleotide phosphodiesterase activity of buffalo sperm midpieces and tails was resolved by gel filtration on Sephadex G-200 into isozymes, phosphodiesterase I and phosphodiesterase II, which were observed to be kinetically different. Phosphodiesterase I and phosphodiesterase II showed different substrate affinities (phosphodiesterase I, K m = 14.5 μM and phosphodiesterase II, K m = 45 μM with cyclic AMP as the substrate). While phosphodiesterase II hydrolysed cyclic GMP as well ( K m = 20 μM), no such activity was obsserved for phosphodiesterase I. Heat inactivation studies on phosphodiesterase II indicated the association of cyclic AMP- and cyclic GMP-phosphodiesterase with a single protein and mixed substrate analysis showed catalysis of two substrates at a single site. The effect of methylxanthines (caffeine and theophylline), adenine nucleotides (ATP and ADP), cyclic GMP, divalent metal atoms (Mg 2+, Mn 2+, Ca 2+) was seen on phosphodiesterase I and phosphodiesterase II. ATP inhibited cyclic AMP hydrolysis by phosphodiesterase I and phosphodiesterase II non-competetively and reduced V to half its value at concentrations between 2–3 mM. Cyclic GMP had no effect on cyclic AMP hydrolysis by phophodiesterase I but inhibited non-competetively that by phosphodiesterase II. At 25 μM concentration of cyclic GMP, V of phosphodiesterase II was reduced to more than one half. Hill plots in the presence of both ATP and cyclic GMP showed no allosteric interactions for phosphodiesterase I and phosphodiesterase II. Mn 2+ stimulated cyclic AMP-phosphodiesterase activity of phosphodiesterase II at lower concentrations but was inhibitory at higher ones. The results are discussed in relation to control of intracellular cyclic AMP levels by phosphodiesterase I and phosphodiesterase II and it is suggested that ATP may serve as the physiological regulator of sperm motility.

Assay and properties of the enzyme catalyzing the biosynthesis of 1- O-alkyl dihydroxyacetone 3-phosphate

The properties of the enzyme alkyl dihydroxyacetone phosphate synthase (1- O-acyl dihydroxyacetone 3-phosphate + ROH → 1- O-alkyl dihydroxyacetone 3-phosphate + fatty acid) in Ehrlich ascites tumor cell microsomes were studied utilizing a new assay procedure. The assay is based on the difference of partition coefficient between alkyl dihydroxyacetone- P and hexadecanol in an alkaline two-phase CHCl 3-methanol-water system. The product of the enzymatic reaction was characterized by different methods as alkyl dihydroxyacetone- P. At optimum concentration of the substrates (acyl dihydroxyacetone- P and hexadecanol), detergents or Mg 2+ and ATP do not stimulate the reaction. The kinetic properties of the reaction were found to be complex due to the stimulation of enzyme activity at low acyl dihydroxyacetone- P concentration but inhibition at the high concentration of the same substrate. The enzyme accepts a variety of long-chain primary alcohols as substrate without much specificity. With different acyl dihydroxyacetone- P, the highest activity was obtained with hexadecanoyl (16:0) dihydroxyacetone- P, with somewhat less activity with longer octadecanoyl (18:0) and much less (40% of 16:0) activity with tetradecanoyl (14:0) dihydroxyacetone- P. Preliminary studies indicate that a Schiffs base intermediate of the enzyme with the keto substrate is probably not formed, since NaBH 4 inhibited the enzyme to the same extent in the presence or absence of acyl dihydroxyacetone- P. Fatty acids inhibit the reaction and an exchange of fatty acid with acyl dihydroxyacetone- P in the presence of the enzyme preparation was observed. Heat-inactivation studies indicate that alkyl dihydroxyacetone- P synthase is probably also catalyzing this fatty acid exchange reaction. Based on these findings, an enzyme-bound dihydroxyacetone- P intermediate is proposed.

Serous ovarian neoplastic amylase (SONA): a potentially useful marker for serous ovarian tumors.

A series of cystic ovarian tumors was studied with regard to amylase activity in the cyst fluid. Large amounts of amylase activity were present only in those patients with endosalpingeal-type lining epithelium (serous ovarian neoplasms). The amylase activity from two such patients was further characterized using G75 Sephadex chromatography, DEAE Sephadex chromatography, agarose gel chromatography, isoelectric focusing, and heat inactivation studies. Serous ovarian neoplastic amylase (SONA) exhibited properties different from amylases of pancreatic and salivary gland origin. Serous ovarian neoplastic amylase (SONA) may be a pancreatic and salivary gland origin. Serous ovarian neoplastic amylase (SONA) may be a useful biochemical marker in diagnosing and managing patients with serous ovarian neoplasms.

Serum and milk alkaline phosphatase in human lactation

Serum and milk alkaline phosphatase (AP) values are reported for the first 16 weeks of lactation. Serum levels at all stages are elevated above normal non-pregnant controls. Sequential milk AP values are reported for the first time. Heat inactivation and phenylalanine inhibition studies would suggest that serum AP in lactation is composed of bone and liver isoenzymes, but it shows an increase in the rate at which enzymatic activity is lost when frozen, indicating that lactation serum AP is largely a different isoenzyme. Electrophoresis demonstrates several AP “slow” bands of unknown origin in lactation serum.

Specificity of porcine liver Galβ(1 → 3)GalNAc-R α(2 → 3) sialyltransferase Sialylation of mucin-type acceptors and ganglioside G M1 in vitro

Porcine liver microsomes are capable of transferring sialic acid from CMP-NeuAc to [ 14C]galactosylated ovine submaxillary asialo-mucin, porcine submaxillary asialo/afuco-mucin and ganglioside G M1. The specificity of the porcine liver sialyltransferase (CMP- N-acetylneuraminate: dgalactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) towards the first acceptor, [ 14C]Gal-GalNAc-protein, was investigated by means of methylation studies on the oligosaccharide chains cleft-off from the sialylated product glycoprotein by β-elimination under reductive conditions. It appeared that sialic acid was transferred solely to position C-3 of galactose residues on Galβ(1 → 3)GalNAc disaccharide units. Transfer to GalNAc residues was completely absent. Competition experiments and heat inactivation studies suggested that the same enzyme also converts ganglioside G M1 to ganglioside G D1a. Therefore, this porcine liver sialyltransferase can be designated as a Galβ(1 → 3)GalNAc-R α(2 → 3) sialyltransferase.