Identification in Tetrahymena pyriformis of 3-hydroxy-3-methyl glutaryl coenzyme a lyase: its purification and properties

The major HMG-CoA utilizing enzyme activity in T. pyriformis has been determined to be HMG-CoA lyase. The enzyme was purified 32-fold to a specific activity of 431 units/mg from a mitochondrial fraction. Sephacryl S-200 chromatography gave an estimated molecular weight of 50,000 daltons for the HMG-CoA lyase. SDS gel electrophoresis revealed two bands stained by Coomassie Blue--a major band of 50,000 daltons and a minor band of 25,000 daltons. The latter is believed to be an impurity in the preparation. The enzyme has a pH optimum of 9.0, is stimulated slightly by sulfhydryl reagents, and requires a divalent cation for maximum activity. The KM for HMG-CoA is 15 microM.     

Decarboxylation of p-Tyrosine: A Potential Source of p-Tyramine in Mammalian Tissues

The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.     

Investigations on myo-inositol-1-phosphate synthase from the wild type and the inositol-dependent mutant of Neurospora crassa.

The inositol-dependent mutant of Neurospora crassa lacks inositol-1-phosphate synthetase activity. This defect can be revorted by the addition of high-molecular DNA isolated from the wild type. To elucidate the biochemical background of inositol dependence, inositol-1-phosphate synthetase was studied. A method has been developed fro the isolation of the enzyme from the wild type strain in 10 mg scale by salt fractionation, gel filtration and ion-exchange chromatography. The specific activity of the purified enzyme is 4750 U/mg protein and its purity has increased about 100-fold. Polyacrylamide gel electrophoresis indicated that, in addition to the main enzymatically active band, several accompanying proteins occur in very small amount. The molecular weight of the enzyme is 225,000 daltons. Probably it consists of four subunits, two with a molecular weight of 64,000 daltons and another two of 50,000 daltons. An enzymatically inactive protein has been isolated from the mutant with the same procedure as that of the enzyme; it migrated at gel electrophoreis similarly to the enzyme. It may be supposed that the isolated protein is the defective enzyme molecule.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

L-dopa decarboxylase in Ceratitis capitata white puparia and human: a comparative study

1. L-DOPA decarboxylase (DDC) from Ceratitis capitata and from human kidney have been purified by the same methodology. 2. Both enzymes show mol. wts of 100,000, consisting of two identical mol. wt subunits and solely decarboxylate L-DOPA. 3. In the presence of 5-hydroxytryptophan (5-HTP) only the DDC activity from human kidney is remarkably reduced. 4. Addition of exogenous coenzyme is essential only for human DDC activity. 5. Polyclonal antibodies, raised against DDC purified from insects or humans, cross-react with both antigens.     

Some aspects on L-dopa decarboxylase and p-tyrosine decarboxylase in the central nervous and peripheral tissues of the American cockroach Periplaneta americana

1. Aromatic amino acid decarboxylase activities toward L-DOPA (L-3,4-dihydroxyphenylalanine), 5-HTP (5-hydroxytryptophan) and p-tyrosine in different tissues of the sclerotized and newly ecdysed cockroach were analyzed. 2. The ratios of enzyme activity with regard to L-DOPA and p-tyrosine varied considerably in the tissues and between the two different growth stages. 3. A DOPA decarboxylase and a p-tyrosine decarboxylase were separated by gel filtration and ion exchange chromatography. 4. The optimal pH requirement for both enzymes was 7.5 with the exception of the one decarboxylating 5-HTP. 5. The molecular weights of the cockroach brain DOPA decarboxylase and tyrosine decarboxylase were estimated to be 120,000 and 100,000, respectively. 6. Unlike the mammalian aromatic amino acid decarboxylase, the cockroach DOPA decarboxylase cannot be activated by a small amount of benzene. 7. An increase of over 50-fold of DOPA decarboxylase activity and a 50% reduction of tyrosine decarboxylase activity in the epidermal tissue of the newly ecdysed animals was observed. 8. In the fully sclerotized cockroach, a reversible endogenous inhibitor(s) of DOPA decarboxylase in the integument was observed, suggesting that the DOPA decarboxylase is suppressed in the epidermal tissues when ecdysis does not occur.     

Characterization of Recombinant Human Aromatic l-Amino Acid Decarboxylase Expressed in COS Cells

The expression vector containing the full-length cDNA of human aromatic L-amino acid decarboxylase (EC 4.1.1.28) was transfected in COS cells by a modified calcium phosphate coprecipitation method. The cells transfected with plasmids that had a true direction of the cDNA gave a major immunoreactive band at 50 kDa. This expressed enzyme catalyzed the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA), L-5-hydroxytryptophan (L-5-HTP) and L-threo-3,4-dihydroxyphenylserine. The optimal pH of the enzyme activity with L-DOPA as a substrate was 6.5, whereas the enzyme had a broad pH optimum when L-5-HTP was used as a substrate. Addition of pyridoxal phosphate to the incubation mixture greatly enhanced the activity for both L-DOPA and L-5-HTP.     

Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.     

Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme

Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 mumol acetylcholine (ACh) formed min-1 mg-1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000-dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000- and 56,000-dalton bands after SDS gel separation of crude extract.     

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and 15-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5,13-dienoic acid, suggesting that the inactivation occurred at the active site of the enzyme. The catalytic inactivation of thromboxane synthase and the greater rate of formation of HHT in thromboxane-synthesizing system may probably play important regulatory roles in the control of thromboxane synthesis.     

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...     

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : I. Induction and Purification

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO(2) produced per minute per milligram protein at pH 8.4 and 30 degrees C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 +/- 300 daltons.     

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human alpha 1-proteinase inhibitor.

Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.     

Polysome-dependent synthesis of embryonic proteins of Artemia salina during cell differentiation and analysis of heme-containing protein.

The polysomes isolated from the different developmental stages of Artemia salina have been translated in a homologous S-30 extract or pH 5 enzyme fraction as well as in a heterologous (wheat germ) pH 5 enzyme fraction. The specific template activity of the polysomes increases linearly up to a stage which is equivalent to about two-thirds of the whole preemergence period and thereafter remains rather constant until the nauplius stage. The specific template activity of the polysomes appears to be dependent on a source of in vitro systems, but not to be dependent on the size of the polysomes used. Moreover, the efficiency of polypeptide chain release from the polysomes in vitro also seems dependent on the developmental stages of the embryos, but in this case it is not influenced by the in vitro system employed. Analysis of the proteins synthesized in vitro by the polyacrylamide gel electrophoresis has revealed that the species and their relative amounts of the proteins are rather specific for each developmental stage analyzed.Heme-containing proteins from the nauplii have been partially purified and analyzed. This heme-protein complex contains two distinct polypeptide chains, MW > 100,000 and MW = 50,000–60,000 daltons, respectively, as the major species as judged by SDS-polyacrylamide gel electrophoresis. During preemergence development, no heme-protein complexes have been detected, but after hatching of the embryos, they have been detected.     

IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.     

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.     

Isolation and properties of superoxide dismutase from human blood plasma]

A procedure for purification of superoxide dismutase (SOD) from human blood plasma has been developed, which includes gel filtration on Ultrogels AcA-34 and AcA-44 (LKB, Sweden). The protein purified from blood plasma is a glycoprotein which is thermostable at 70-80 degrees C. The molecular mass of the protein determined immediately after gel filtration is approximately 147,000 daltons. A comparative analysis of effects on the SOD activity of plasma and erythrocytes of compounds capable of forming chelating complexes with metals within the enzyme active center has been carried out. The purified enzyme differs by its physico-chemical characteristics from cytosolic Cu,Zn-SOD and pertains to a new class of SOD, the so-called extracellular SOD, detected in some biological fluids.