Active Sulfhydryl Group Research Articles

Low back pain: justification of treatment standards (literature review)

Low back pain: justification of treatment standards (literature review)

Effect of low-frequency alternating magnetic fields on the physicochemical, conformational and rheological properties of myofibrillar protein after iterative freeze-thaw cycles

In this work, the effects of low-frequency alternating magnetic fields (LF-AMF) on the physicochemical, conformational, and functional characteristics of myofibrillar protein (MP) after iterative freeze-thaw (FT) cycles were explored. With the increasing LF-AMF treatment time, the solubility, active sulfhydryl groups, surface hydrophobicity, emulsifiability, and emulsion stability of MP after five FT cycles evidently elevated and then declined, and the peak value was obtained at 3 h. Conversely, the moderate LF-AMF treatment time can significantly reduce the average particle size, carbonyl content, and endogenous fluorescence intensity of MP. The rheology results showed that various LF-AMF treatment times would elevate the G' value of MP after iterative FT cycles. The FTIR spectroscopy results suggested that LF-AMF influenced the secondary structure of MP after multiple FT cycles, resulting in a depression in α-helix content and an increment in β-folding proportion. Moreover, LF-AMF treatment induced the gradually lighter and wider myosin heavy chain bands of MP, implying that LF-AMF accelerated the degradation of macromolecular aggregates. Therefore, the LF-AMF treatment efficaciously ameliorates the structural and functional deterioration of MP after iterative FT cycles and could be used as a potential quality-improving technology in the frozen meat industry.

Conformation changes and emulsifying properties of myofibrillar proteins in water: Effects of electrostatic interaction with chitosan

Great interests have been attracted toward muscle protein in a water-soluble state with improved functionality for further designing meat protein fortified low-salt functional foods. In the present study, electrostatic interaction of chitosan (CH) with myofibrillar proteins (MP) in water aqueous solution was investigated, and the linked structure changes and emulsion stabilization of MP were studied. Results showed that the electrostatic interaction inhibited MP aggregation, and smaller particle size complexes were formed at pH 6.0, leading to the loss of β-sheet contents and recovery of α-helix contents with decreasing MP/CH mixing ratio (5:1 and 1:1). The tertiary structure confirmed the conformation changes of MP in which more hydrophobic groups and active sulfhydryl groups were exposed (P < 0.05), and the fluorescence was also quenched. With decreasing mixing ratio, the droplet size of emulsion decreased (P < 0.05), while the absorbed protein content increased (P < 0.05). After 7 d of storage, complex at a ratio of 1:1 displayed desirable emulsion stability, which could be due to the improved emulsifying capacity, enhanced electrostatic repulsion and steric effects. These findings provide a better understanding of conformation changes of MP in water aqueous solution induced by electrostatic interactions at mild acidic pH and help to fabricate stable protein/polysaccharide emulsification systems for further developing meat protein-based functional food to deliver health.

Effect of Ultrasound-Assisted Sodium Bicarbonate Treatment on Aggregation and Conformation of Reduced-Salt Pork Myofibrillar Protein.

To study the effects of an ultrasound (0, 30, and 60 min) and sodium bicarbonate (0% and 0.2%) combination on the reduced-salt pork myofibrillar protein, the changes in pH, turbidity, aggregation, and conformation were investigated. After the ultrasound-assisted sodium bicarbonate treatment, the pH increased by 0.80 units, the absolute value of Zeta potential, hydrophobic force, and active sulfhydryl group significantly increased (p &lt; 0.05), and the turbidity and particle size significantly decreased (p &lt; 0.05). Meanwhile, the fluorescence intensity decreased from 894 to 623, and the fluorescence peak showed a significant redshift, which indicated that the ultrasound-assisted sodium bicarbonate treatment exposed the non-polarity of the microenvironment in which the fluorescence emission group was located, leading to the microenvironment and protein structure of myofibrillar tryptophan being changed. Overall, an ultrasound-assisted sodium bicarbonate treatment could significantly improve pork myofibrillar protein solubility and change the protein structure under a reduced-salt environment.

Effects of Different Quick-freezing Methods on Protein Properties of Largemouth Bass( Lateolabrax japonicus )

In this paper, largemouth bass is used as raw material to study the effects of different cooling rates(liquid nitrogen quick freezing(1.81 ℃), freezing liquid quick freezing(0.15 ℃), flat plate quick freezing(0.14 ℃)) in freezing storage(0, 1, 2, 4, 12, 24 weeks), the influence of the physical and chemical properties of sea bass protein, by measuring the salt-soluble protein, sulfhydryl group, carbonyl group, Ca2+-ATPase activity content, surface hydrophobicity, endogenous fluorescence spectrum and protein composition changes in the fish meat, and using double factor variance and correlation analysis study quick-freezing method and storage time to investigate the degeneration of fish protein after freezing. The results showed that with the passage of frozen storage time, the value of salt-soluble protein showed a downward trend: The plate group and liquid nitrogen group had the lowest and the highest at the end of the frozen storage, respectively; the activity values of sulfhydryl and Ca2+-ATPase both increased first and then decreased. The endogenous fluorescence intensity of fibrin increased, resulting in a blue shift phenomenon, and the value of myofibrillar protein carbonyl and surface hydrophobicity increased significantly(P<0.05). The results of SDS-PAGE electrophoresis showed that myofibrillar protein was degraded during the freezing period, while the liquid nitrogen group had a faster cooling rate and a slower degree of protein degradation, while the plate group was the opposite. The volume of ice crystals formed by quick freezing of liquid nitrogen was similar to the distribution of water in the raw material, which was conducive to storage. Compared with liquid nitrogen quick freezing, the time for the formation of the maximum ice crystal zone in the quick freezing of liquid nitrogen was longer than that of liquid nitrogen quick freezing and shorter than that of flat quick freezing, and there was not much difference between the two. The correlation analysis between the two-factor variance and the indicators showed that the quick freezing method had a significant effect on the active sulfhydryl group and the maximum fluorescence intensity of myofibril protein(P<0.05). The freezing time was the main factor affecting the protein of the sea bass. The oxidative modification of amino acid side chain groups in myofibrillar protein was the main factor causing the degradation or aggregation of protein.

Structural, functional, and sensorial properties of protein isolate produced from salmon, cod, and herring by-products

Nutritional, structural, functional, and sensorial properties of protein isolate developed from salmon (Salmo salar), cod (Gadus morhua), and herring (Clupea harengus) by-products using the pH-shift method was studied. Function of the proteins in an emulsion system in terms of viscoelastic properties was also evaluated. Regardless of origin, the proteins showed satisfying nutritional value as reflected in their high essential amino acid content. The proteins contained significantly (p < 0.05) higher proportion of active sulfhydryl groups and surface hydrophobicity compared to whey and egg white protein reflecting conformational changes caused by the pH-shift process. Solubility, emulsion, and foaming capacity of the proteins showed a trend similar to soy protein and dependent on their origin. Cod protein had better emulsion and foaming capacity than salmon and herring proteins which was in line with its high surface hydrophobicity and myosin heavy chain content. Emulsions developed from cod and salmon proteins showed substantially better viscoelastic properties, with higher stability and viscosity compared to herring protein emulsions. Cod protein scored low for sensorial attributes related to lipid oxidation while herring protein showed high levels of fishy and rancid flavor and odor. Altogether, results showed that the proteins from fish filleting by-products have potential to be used as food ingredients, but their application would be governed by their origin and sensorial properties.

Evaluation of the Future Atherosclerotic Heart Disease with Oxidative Stress and Carotid Artery Intima Media Thickness in Gestational Diabetes Mellitus

Background and objective. In this study our aim was to evaluate paraoxonase (PON1) activity and free sulfhydryl groups (–SH) as antioxidative parameters and lipid hydroperoxide (LOOH) as oxidative parameter in the serum of women with gestational diabetes mellitus (GDM) and determine their relation with the degree of subclinical atherosclerosis. Material and methods. Serum samples from 39 pregnant women complicated with GDM and 40 healthy pregnant women were collected for the analysis of oxidative markers. Common carotid artery intima media thickness (CIMT) was measured for both groups to assess future atherosclerotic heart disease risk. PON1 activity and –SH were measured spectrophotometrically. LOOH levels were measured by ferrous oxidation with a xylenol orange assay. Results. CIMT and LOOH levels were significantly higher (p = 0.01, p < 0.001, respectively) in GDM group compared to controls, whereas PON1 and –SH levels were significantly lower (p < 0.001 for both). CIMT values were significantly correlated with body mass index (BMI), 50 g oral glucose tolerance test (OGTT), and mean arterial blood pressure (MABP) (p = 0.003, p = 0.02, and p = 0.03, respectively). However, there was no correlation between CIMT and oxidative markers. Conclusions. Increased levels of LOOH and decreased levels of PON1 and –SH showed disturbance of antioxidative mechanisms in GDM. These changes were associated with increased BMI and MABP which may be relevant to GDM pathophysiology. Furthermore, increased CIMT values in GDM compared to healthy controls designate increased risk of future atherosclerotic heart disease.

Development of a spiral piezoelectric immunosensor based on thiol self-assembled monolayers for the detection of insulin

A spiral piezoelectric (Pz) immunosensor based on thiol self-assembled monolayers (SAMs) and fabricated with a screw clamp apparatus was developed for quantitative detection of insulin in serum. Anti-human insulin antibody was first modified with sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP) to graft the active sulfhydryl groups into antibody molecules. Then, modified antibody molecules were assembled on a gold electrode via covalent bonding to form SAMs as the recognition element of the insulin immunosensor. The response characteristics of the Pz immunosensor, including buffer solution, time-costs, reproducibility and specificity, were also investigated in detail. Coating a Au electrode with SAMs gave better results within 40 min, with a detection range of 2.5 to 80 mIU/L and a coefficient of variance (CV) less than 5%. The stability and reusability of the immunosensor was improved with a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized antibody. A comparison study revealed that analytical results of insulin in samples obtained with this immunosensor were in good agreement with those given by the radioimmunoassay. Thus, the proposed Pz immunosensor provided a rapid, sensitive, specific, reusable and reliable alternative for the detection of insulin in clinical laboratory.

1,1,2-Tris-organoselenide alkene derivatives, but not 1,2-bis-organoselenide alkene derivatives, inhibited δ-aminolevulinate dehydratase activity from human erythrocytic cells in vitro

Organochalcogens are important intermediates and useful reagents in organic synthesis. Recent data from our laboratory demonstrated that bis and tris-selenide alkene derivatives are attractive synthetic targets because of their chemio-, regio- and stereo-selective reactions. Since the erythrocytic δ-aminolevulinate dehydratase (δ-ALA-D) activity could be an important indicator of toxicity, this report investigated bis and tris-selenide alkene derivatives effects on blood δ-ALA-D in vitro. To investigate the mechanisms by which these compounds inhibit human blood δ-ALA-D activity, a thiol reducing agent or zinc chloride were used. 1,2-Bis-selenide alkene derivatives 1a (R = 4-MeOC 6H 4), 1b (R = 4-ClC 6H 4) and 1c (R = 2,4,6-Me 3C 6H 2) did not inhibit human blood δ-ALA-D activity. 1,1,2-Tris-selenide alkene derivative 2a (R = C 6H 5) was the most potent δ-ALA-D inhibitor. Compounds 2b (R = 4-MeOC 6H 4) and 2c (R = 4-ClC 6H 4) displayed similar inhibitory potency towards δ-ALA-D activity. Dithiothreitol, a hydrophobic SH-reducing agent, was able to restore and to protect δ-ALA-D activity inhibited by tris-selenide alkene derivatives. Conversely, ZnCl 2 did not alter the enzyme inhibition induced by tris-selenide alkene derivatives. From these findings we suggest that 1,1,2-tris-selenide alkene derivatives inhibited δ-ALA-D activity by an interaction with essential sulfhydryl groups for the enzyme activity.

Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2- Arachidonoyl-Glycerol in Rat Cerebellar Membranes

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.

Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species

Hypertension and coronary artery disease are intimately connected. The migration of circulating monocytes into the subendothelial occurs through the expression of some adhesion molecules on endothelial cells. The nuclear factor (NF)-κB, a redox-sensitive element, plays a key role in adhesion molecule gene induction. In this study we have compared the effects of two different angiotensin converting enzyme (ACE) inhibitors, one possessing an active sulfhydryl group (zofenopril) and one lacking this group (enalapril) on the cellular redox state (monitored by measuring intracellular reactive oxygen species and thiol status), expression of adhesion molecules, and activation of NF-κB in human umbilical vein endothelial cells (HUVECs). Zofenoprilat, the active form of zofenopril, significantly and dose dependently reduced the intracellular reactive oxygen species (ROS) and superoxide formation induced by oxidized low-density lipoprotein (ox-LDL) ( P < .001) and tumor necrosis factor-α (TNF-α) ( P < .001). Enalaprilat, the active form of enalapril, was ineffective. Zofenoprilat but not enalaprilat also decreased the consumption of the intracellular GSH induced by ox-LDL ( P < .01) and TNF-α ( P < .01). Although zofenoprilat significantly and dose dependently reduced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin induced by ox-LDL ( P < .01) and TNF-α ( P < .01) on HUVECs, enalaprilat did not. Ox-LDL and TNF-α increased the activation of NF-κB and the preincubation of HUVECs with zofenoprilat, but not with enalaprilat, dose dependently reduced its activation ( P < .001). The conclusion is that the sulfhydryl (SH)-containing ACE inhibitors may be useful in inhibiting foam cell formation and thus slow the development of atherosclerosis.

5 β-Scymnol sulfotransferases from the liver of two Australian ray species

The tissue distribution of sterol sulfotransferase activity was investigated within the ray species Trygonorrhina fasciata and Trygonoptera species. The liver and kidney cytosolic fractions of each species were found to contain an enzyme that catalyzed the transfer of the sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to the elasmobranch bile alcohol 5 β-scymnol. The liver is the major site of bile salt biosynthesis and detoxication, with the kidney possibly playing a secondary role. The two ray species liver sulfotransferases were partially purified by column chromatography and characterized. The enzymic properties of the partially purified liver sulfotransferases from the T. fasciata and Trygonoptera sp. ray species were similar in character. They followed Michaelis–Menten kinetics and were inhibited by the substrate 5 β-scymnol and the reaction product adenosine-3′,5′-diphosphate. The enzymes were activated by the addition of magnesium ions, but had no absolute requirement for these ions; however, they were found to require sulfhydryl groups for activity. The steroid alcohols 5 β-scymnol and 5 α-cyprinol were both substrates for the sulfation reaction and the enzymes showed pH optimums of about 6.5, with a range of 5.5–7.5 and a temperature range from 22 to 37°C. This is the first report concerning sulfotransferase activity in ray species.

Release of a newly-identified cysteine protease, tetrain, from Tetrahymena into culture medium during the cell growth.

Protease activity in the culture medium of Tetrahymena pyriformis markedly increased during the growth of the ciliate. The protease activity in the culture medium was purified by sequential column chromatographies. The purified protease had an apparent molecular mass of 28 kDa. N-terminal amino acid sequencing analysis suggested that the protease is a mature form of cysteine protease. Requirements of free sulfhydryl groups for activity and sensitivity to N-tosyl-L-phenylalanine chloromethyl ketone and Na-p-tosyl-L-lysine chloromethyl ketone also indicated that the protease is a member of the papain family of cysteine proteases. The protease was designated as tetrain. Immunoblotting analyses showed that tetrain was present in higher amount in the culture medium in the stationary phase than in the logarithmic phase. Tetrain has high activities at neutral to alkaline pH values. This suggests that tetrain has functional roles in the culture medium in the stationary phase, because the pH of the culture medium became alkaline with the progress of Tetrahymena growth.

A Novel Invertase from a Thermophilic FungusThermomyces lanuginosus:Its Requirement of Thiol and Protein for Activation

Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungus,Thermomyces lanuginosus,was unusually unstable bothin vivoandin vitro.The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b)N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction. In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds. Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation. A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called “PRIA” for theprotein which helps inrestoringinvertaseactivity. The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form. A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations. The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source. The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry.

Identification and Distribution of Peptide:N-Glycanase (PNGase) in Mouse Organs

A wide occurrence of peptide:N-glycanase (PNGase) in mouse organs was demonstrated. PNGase activities were determined using 14C-labeled fetuin glycopeptide I as a substrate by a newly improved enzyme assay based on the paper chromatographic and paper electrophoretic analyses. PNGase activities were detected in both soluble and membranous (or particulate) fractions, although the levels of the activities were different from organ to organ. Soluble PNGases were partially purified from brain, liver, kidney, and spleen by TSK butyl-Toyopearl 650 M hydrophobicity chromatography and characterized for enzymatic properties. The soluble enzymes were found to share the following properties: (a) high hydrophobicity; (b) sensitivity to metal cations such as Zn2+, Cu2+ and Fe3+; and (c) requirement of sulfhydryl group(s) for enzyme activity. Notably, soluble PNGases were unable to degrade glycoasparagine substrates and the optimal pH was near 7.0, suggesting that they were not lysosomal enzymes, but perhaps being involved in basic biological processes in certain intracellular nonlysosomal events. All of these enzymatic properties found for mouse organ-derived PNGases were the same as those recently found for L-929 PNGase that was highly purified as a soluble enzyme from mouse fibroblast L-929 cells (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1994) J. Biol Chem. 269, 17611-17618).

Enzymic sulfation of bile salts. Partial purification and characterization of an enzyme from the liver of the shark Heterodontus portusjacksoni that catalyses the sulfation of the shark bile steroid 5β-scymnol

An enzyme system which catalyses the transfer of the sulfate group from 3′-phosphoadenosine-5′-phosphosulfate to the bile steroid 5β-scymnol has been isolated and characterized from the liver of the shark Heterodontus portusjacksoni (Meyer 1793). The enzyme is present in the cytosol fraction of liver cells. It was partially purified by hydroxylapatite chromatography, molecular sizing by G100-Sephadex and isoelectrofocusing electrophoresis. The apparent K m value for 3′-phosphoadenosine-5′-phosphosulfate was 4μM and that for 5β-scymnol, 14μM. The enzyme activity is inhibited by iodoacetate and p-chloromercuribenzoate indicating the possible requirement of a sulfhydryl group for activity. The molecular weight of the enzyme was estimated to be 40 kDa by gel filtration. This was verified by running the partially purified material on a native gel and electrophoretically separating two major bands corresponding to molecular weights of 40 and 45 kDa, respectively. Isoelectric focusing of the purified material resulted in two major bands with pI values of 5.0 and 5.85. Enzymatic activity was found to be optimal at a pH of 6.5 with little activity recorded at pH 5.0 and 8.0.

On the mechanism of inactivation of active papain by ascorbic acid in the presence of cupric ions.

An inactivation mechanism of active papain (EC 3.4.22.2) by the Cu(2+)-ascorbic acid (AsA) system was examined. Incubation of active papain, which contains an active sulfhydryl (SH) group, with the Cu(2+)-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. The enzyme was not inactivated at a molar ratio of enzyme to Cu2+ of 1:< 1, whereas at a molar ratio of 1:1-2, the extent of inactivation showed the same dependence on the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. The degree of inactivation was dependent on the decrease in SH content of the enzyme. Catalase at a low concentration partially protected the enzyme from inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and AsA from oxidation. The present results suggest that an additional function of H2O2, besides producing hydroxyl radicals (.OH), is to promote the conversion of Cu+ into Cu2+, and that an active SH group of papain is site-specifically modified by the .OH, resulting in inactivation of the enzyme.

Post-transcriptional regulation of tissue-specific isoforms. A bovine cytosolic RNA-binding protein, COLBP, associates with messenger RNA encoding the liver-form isopeptides of cytochrome c oxidase

Regulation of the liver isopeptides of bovine cytochrome c oxidase is reported to be post-transcriptional. Extensive interspecies sequence homologies exist in the 3'-untranslated regions for transcripts encoding these liver isoforms, suggesting that these regions may be involved in mediating regulation of mRNA expression. To explore this possibility, several bovine tissue homogenates were assayed for any trans-acting factors that recognized the transcript encoding the liver isoform of subunit VIII (BCOL8). Such a protein factor (COLBP: cytochrome c oxidase L-form transcript-binding protein) was identified in liver, kidney, and lung tissue and was shown to require free sulfhydryl groups for activity. No binding activity, however, was found in muscle-type homogenates. Furthermore, this binding protein also recognized the subunit VIIa-liver transcript but was unable to associate with the mRNA encoding the heart isoform of subunit VIII. Intriguingly, the tissue-specific distribution of COLBP activity parallels the presence of the liver isopeptides in the mature oxidase complex. It is therefore suggested that COLBP may mediate the tissue-specific regulation of cytochrome c oxidase liver isoform mRNA expression.

Properties of [Ca2+ + Mg2+]-Adenosine Triphosphatases in the Golgi Apparatus and Microsomes of the Lactating Mammary Glands of Cows

The [Ca2+ + Mg2+]-ATPase activity of bovine lactating mammary gland is associated with membranes. This study compares the ATPase activity in microsomal membranes to that of the Golgi apparatus. The enzyme activity in both fractions hydrolyzed Ca2+-ATP and Mg2+-ATP. The ATPase activities were inhibited by ρ-chloromercuribenzoate, indicating the involvement of a sulfhydryl group for activity. Although calmodulin had no effect on the ATPase activities of the two fractions, calmodulin antagonists (chlorpromazine, fluphenazine, and trifluoperazine) were inhibitory. Strong inhibitors of the ATPase activities were vanadate, dicclohexylcarbodiimide, La3+, and Zn2+. There were some differences in the activities from two membrane fractions. Although both fractions could hydrolyze all of the triphosphonucleotides, cytidine-5’-triphosphate and uridine-5’-triphosphate were poor substrates for the Golgi enzyme. Detergents diminished the activity of the microsomal enzyme to a much greater extent than the ATPase of the Golgi apparatus. Thus, the intact membrane may be more critical to microsomal activity. The role of these enzymes in Ca2+ accumulation in milk is discussed.

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and physical properties.

Compared to other tRNAs all known histidine tRNAs have the unique feature of possessing an additional nucleotide at their 5' end. It is usually a guanosine residue but not in bacteriophage T5 tRNA which carries an additional uridine. The additional nucleotide is not encoded in eukaryotic histidine tRNA genes but is added in a post-transcriptional modification reaction (Cooley, L., Appel, B., and Söll, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6475-6479) by histidine tRNA guanylyltransferase (tRNAHis guanylyltransferase). Here we report the purification of this enzyme from Saccharomyces cerevisiae and the determination of some of its physical properties. Six different steps including Polymin P precipitation, chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, ATP-agarose, and gel filtration on Superose 12 were employed for the purification of the guanylyltransferase from an S-100 extract. A Stokes radius of 46.5 +/- 0.5 A and a sedimentation coefficient (S20,w) of 7.8 +/- 0.2 were determined by gel filtration and rate zonal sedimentation, respectively. A relative molecular weight (Mr) of approximately 120,000 was calculated for the purified native enzyme. The Mr of the denatured protein is approximately 58,000 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate a homodimeric (alpha 2) structure for the enzyme. Among all acceptor RNAs in unfractionated tRNA only tRNAHis is a substrate for the purified guanylyltransferase. The reaction requires ATP, a guanosine substrate, and a divalent metal ion. Treatment of guanylyltransferase with 5,5'-dithiobis(2-nitrobenzoic)-acid abolishes activity; this suggests the importance of sulfhydryl groups for enzymatic activity. The enzyme shows discrimination among different histidine tRNA species; tRNA from plant and prokaryotes are better substrates than mammalian and insect tRNAs.