Isolation and characterization of ornithine transcarbamylase from normal human liver.

We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue.     

Chorismate mutase isoenzymes from Sorghum bicolor: immunological characterization

Highly purified fractions of chorismate mutase 1 and 2 from etiolated seedlings of Sorghum bicolor were used as the antigen for antibody production in BALB/c mice. Tests for antigen-antibody complex formation were made by immunodiffusion, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). These tests indicated the presence of specific antibodies for each isoenzyme in their antisera. However, in the same tests, no cross-reaction was found between chorismate mutase 1 and 2 and their antisera. This indicates no immunological similarity between the two isoenzymes of chorismate mutase from sorghum.     

Catalytic properties of human liver alcohol dehydrogenase isoenzymes

Human liver alcohol dehydrogenase (ADH) exists in multiple molecular forms which arise from the association of eight different types of subunits, alpha, beta 1, beta 2, beta 3, gamma 1, gamma 2, pi, and chi, into active dimeric molecules. A genetic model accounts for this multiplicity as products of five gene loci, ADH1 through ADH5. Polymorphism occurs at two loci, ADH2 and ADH3, which encode the beta and gamma subunits. All of the known homodimeric and heterodimeric isoenzymes have been isolated and purified to homogeneity. Analysis of the steady-state kinetic properties and substrate and inhibitor specificities has shown substantial differences in the catalytic properties of the isoenzymes. For example, the Km values for NAD+ and ethanol vary as much as 1,000-fold among the isoenzymes. Some of the differences in catalytic properties can be related to specific amino acid substitutions in the ADH isoenzymes.     

Physico-chemical and kinetic properties of hexokinase isoenzymes from human normal and tumor tissues]

Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000.     

Cathepsin D. Purification of isoenzymes from human and chicken liver

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, alpha,beta and gamma, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified beta and gamma isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.     

Protein disulphide-isomerase from human placenta and rat liver. Purification and immunological characterization with monoclonal antibodies.

The purification of human placenta and rat liver protein disulphide-isomerase (PDI, EC 5.3.4.1) and the production of a panel of monoclonal antibodies against these proteins are described. The physical and enzymic properties of human PDI and rat PDI were similar; immunological characterization revealed the presence of unique, as well as shared, antigenic determinants. Although purified rat liver PDI was present as three forms differing slightly in Mr value, evidence was presented that the multiple forms represent proteolytic degradation products of a single 59,000-Mr species. Purified human PDI had an apparent Mr of 61,200. Two of the monoclonal antibodies against human PDI partially inactivated the enzyme, and one of these in indirect immunoprecipitation led to the precipitation of all glutathione:insulin transhydrogenase activity from a crude extract of human placenta. Results of immunofluorescence experiments with HT-29 human colon carcinoma cells were consistent with localization of PDI in the nuclear membrane and cell cytoplasm.     

The molecular defect leading to Fabry disease: structure of human alpha-galactosidase

Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme alpha-galactosidase (alpha-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human alpha-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (beta/alpha)8 domain with the active site and an antiparallel beta domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of alpha-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in alpha-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human alpha-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.     

Alkaline phosphates from human milk. Comparison with isoenzymes from placenta and liver.

Alkaline phosphatase was partially purified from human milk and its antigenic, functional and structural properties were characterized. By immunochemical and enzymic criteria, the enzyme resembled the alkaline phosphatase isoenzyme found in human liver. Two-dimensional electrophoretic analysis showed that the milk enzyme differed from the liver both in subunit molecular weight and in isoelectric point. These differences were shown to result from variation in sialic acid content.     

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.     

Purification and characterization of alpha-galactosidase from watermelon

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.     

Alkaline phosphatase isoenzymes in human kidney and urine,immunohistochemical, immunological and biochemical characterization

Summary Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%–4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%–40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.     

Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver.

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.     

Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.