The isolation and partial characterization of catalase and a peroxidase active fraction from human white adipose tissue.

1. A procedure is described for the purification of catalase and a peroxidase active fraction from human white adipose tissue. 2. Gel electrophoresis on SDS-PAGE revealed relative molecular masses of 202,900 and 208,600 for the active catalase and peroxidase molecules respectively (nonreducing conditions), as compared to 56,800 and 49,800 for the monomers under reducing conditions, thus indicating the likelihood of tetramers in the intact state. 3. The two purified enzymes differ with regard to pH optima (5-9 for catalase and 3 for peroxidase), temperature stability (up to 50 degrees C for catalase and 70 degrees C for peroxidase) and Km values towards H2O2 (38.9 mM for catalase and 7.69 mM for peroxidase, which was also active in oxidizing a number of o-dihydricphenols as second substrates). 4. The catalase enzyme showed uncompetitive inhibition by the irreversible inhibitor 3-amino-1,2,4-triazole (AT), Ki = 5.4 mM.     

Rapid isolation of plant peroxidase. Purification of peroxidase a from Petunia

A rapid isolation procedure was developed for purification of peroxidase a from Petunia hybrida . Rapid isolation was possible since about 15% of the extracellular protein from stem tissue obtained by vacuum infiltration followed by centrifugation of the tissue represents peroxidase. Purification of peroxidase a from intercellular fluid was achieved by two acetone precipitation steps followed by DEAE-cellulose chromatography.
Three different forms of peroxidase were eluted from DEAE-cellulose at different NaCl concentrations. Isoelectric focusing showed, however, a pI of 3.8 for all three forms of peroxidase a . Only part of the peroxidase a enzymes bound to Concanavalin A indicating heterogeneity in the carbohydrate part. Homology of peroxidase a to the peroxidase G₁ group from tobacco is discussed.     

The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.     

Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional chinese medicines

There is considerable interest in the isolation of more potent antioxidant compounds to treat diseases involving oxidative stress. Thirty-three traditional Chinese medicine (TCM) extracts were examined for their antioxidant activity using the 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonate] (ABTS) assay. Five extracts with high activity (Cratoxylum cochinchinense, Cortex magnoliae officinalis, Psoralea corylifolia L, Curculigo orchioides Gaertn, and Glycyrrhiza uralensis Fisch.) were selected for further characterization. C. cochinchinense outperformed other extracts in most of the assays tested except phospholipid peroxidation inhibition, where P. corylifolia L showed higher activity. C. cochinchinense was particularly potent in inhibiting the formation of advanced glycation end products on proteins and strongly inhibited hypochlorous acid-induced DNA damage. We attempted to isolate the active ingredients from C. cochinchinense and obtained an extract (YCT) containing at least 90% mangiferin as identified by HPLC and mass spectrometry. However, YCT showed significantly higher activity in assays of phospholipid peroxidation, inhibition of protein glycation, and superoxide (O₂^√−) and peroxynitrite (ONOO⁻) scavenging, as compared with mangiferin, suggesting that the nonmangiferin constituents of YCT contribute to its additional antioxidant activities.     

The isolation and properties of mitochondria from rat pancreas

A technique for the isolation of functional rat pancreatic mitochondria is described. The resultant mitochondrial preparations contained oligomycin-insensitive Mg²⁺-ATPase activity and coupled respiration could only be demonstrated in the absence of Mg²⁺ and in the presence of EDTA.     

Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus

The cDNA clone lambda ML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus, Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the lambda ML-1 DNA encodes the lignin peroxidase H8.     

The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.     

Purification and properties of peroxidase from Nitrosomonas europaea

Peroxidase from the obligate chemosynthetic bacterium Nitrosomonas europaea was purified 1,500-fold, and its properties were examined. The enzyme had a molecular weight of 53,000 and exhibited characteristic absorption maxima at 410, 524, and 558 mmu. The optimal pH and temperature were 7.5 and 44 C, respectively. The peroxidase reaction had an energy of activation of 5,850 cal/mole and required a primary substrate (H(2)O(2)) concentration of 7 x 10(-6)m to proceed at half maximal velocity (K(m)). Reduced cytochrome, c,p-phenylenediamine and pyrogallol acted as hydrogen donors to the purified peroxidase-H(2)O(2) complex. Conditions most suitable for the chemical oxidation of ammonium by H(2)O(2) were determined. The reaction was rapid and produced nitrite but no nitrate. Hydroxylamine was not detected as an intermediate, but it could substitute for ammonium in the system. Neither the rate nor the extent of these reactions was influenced by purified peroxidase, and no evidence was obtained to support a conclusion that the enzyme performs a vital role in the transformation of ammonium to nitrite by N. europaea.     

The isolation and properties of collagenolytic proteases from crab hepatopancreas

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp. collagenases. In contrast with microbial collagenases the crab enzymes are good general proteases, able to cleave standard synthetic and protein substrates and possess a chymotrypsin-, trypsin- and elastase-like specificity. N-Terminal sequence analysis revealed that crab collagenolytic proteases had evolved from a trypsin-like ancestor. Crab proteases, structurally belonging to the trypsin-like enzymes, nevertheless, possess the unique ability, among this class of enzymes, to cleave the native insoluble collagen. It seems that crab collagenolytic proteases and true metalloenzyme vertebrate and microbial collagenases have certain common structural features particularly in the regions of their substrate binding site.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.