Evidence that tartrate-resistant acid phosphatases from osteoclastomas and hairy cell leukemia spleen are members of a multigene family.

1. Osteoclasts and hairy cell leukemia spleen both contain large amounts of a band 5-tartrate-resistant acid phosphatase (TrACP). 2. We have recently purified to homogeneity a band 5 TrACP from human osteoclastomas and two isoforms of band 5 TrACP (5a and 5b) from the spleen of a patient with hairy cell leukemia. 3. Although the N-terminal amino acid sequences and the apparent molecular weights of the osteoclastoma, hairy cell leukemia spleen TrACPs were identical, there were several differences in the physical and biochemical properties between the three isoenzymes. 4. Based on these findings, it is concluded that these isoenzymes are different enzymes, but that they could have originated from a similar ancestral gene. 5. It is proposed that the osteoclastoma and hairy cell leukemia band 5 TrACPs are members of a multigene family.     

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.     

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.     

Detection of tartrate-resistant isoenzyme 5 of acid phosphatase in the white blood cells by disc electrophoresis in a case of hairy cell leukemia

In a case of hairy cell leukemia the evidence of tartrate-resistant isoenzyme 5 of phosphatase (TRISP 5) developed to findings important for the diagnosis. The evidence was achieved by means of disc electrophoresis by separating the cell lysate of white blood cells. The percentage of TRISP 5 in the total activity of acid phosphatase in leukocytes amounted to 17.6%. In cytochemical respect the evidence of TRISP 5 proved to be negative. The classification of these findings and their differential-diagnostic significance are discussed.     

Purification and N-terminal amino acid sequence of the human T cell surface antigen T8

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.     

N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture

Summary A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium (1); that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin. The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis (2), which claims that cancer cells produce and respond to their own growth factors (3). The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium. It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts. LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC. The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH₂-terminal sequence analysis. The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis. The 30 NH₂-terminal residues of LGF-I are the same as that of ubiquitin. Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600. In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure (3), and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway (4). However, its physiological significance has not yet been fully resolved. Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested. The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH₂-terminal end. This work was supported by funds obtained under the Research and Development Project of Basic Technologies for Future Industries from the Ministry of International Trade and Industry of Japan. Editor’s Statement Identification of sequences identical to ubiquitin associated with the leukemia-derived growth factor described in this report is particularly intriguing considering recent reports of association of ubiquitin with surface membrane receptors of lymphocytes and fibroblasts. References: SCIENCE vol. 231, pgs, 823–829; NATURE vol. 323, pgs 226–232, 1986. David W. Barnes     

Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.     

Purification and properties of two acid phosphatases from midgut glands of abalone Haliotis discus.

Midgut glands of abalone Haliotis discus contained two acid phosphatases [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] separable by phosphocellulose column chromatography. They were designated as acid phosphatases I and II in order of elution and were purified 99- and 290-fold, respectively. Purified acid phosphatase II was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The substrate specificity of acid phosphatase I was narrow, whereas that of acid phosphatase II was broad. Good substrates for acid phosphatase I included p-nitrophenyl phosphate, phosphoenolpyruvate, inorganic pyrophosphate, and nucleoside di- and triphosphates. The acid phosphatases did not require any metal ion for maximum activity and were inhibited by Zn2+, Cu2+ and Hg2+. Fluoride and arsenate were potent inhibitors of both enzymes. The pH optima of acid phosphatases I and II were 5.9 and 5.5, respectively. The molecular weights of acid phosphatases I and II were estimated to be 28,000 and 100,000, respectively, by gel filtration on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that acid phosphatase II consists of two identical subunits.     

Purification of carboxyl ester lipase from human pancreas and the amino acid sequence of the N-terminal region

A procedure for the purification of carboxyl ester lipase from human pancreas has been developed. The determined N-terminal 10 amino acid residues of the purified enzyme, NH2-Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-Glu-Gly, was identical to the terminal of human milk bile salt-activated lipase. The human pancreatic carboxyl ester lipase has an apparent molecular weight slightly smaller than that of human milk bile salt-activated lipase (105,000 vs 125,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, it is possible that the human pancreatic carboxyl ester lipase and human milk bile salt-activated lipase could be produced by the same gene by a different splice or post-translational modification. Alternatively, they could simply be the products of two closely related but separate genes.     

Purification and characterization of two phosphorylase phosphatases from rabbit liver

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.     

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.     

Purification of acid ribonucleases from bovine spleen

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.     

Purification and N-terminal amino acid sequence of fructose-6-phosphate phosphoketolase from Bifidobacterium longum BB536

AIMS: The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 4.1.2.22.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536). METHODS AND RESULTS: A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 +/- 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 +/- 1 kDa and 59 +/- 0.5 kDa, as determined by SDS-PAGE. CONCLUSION: The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.     

Purification and N-terminal amino acid sequence of solanapyrone synthase, a natural Diels-Alderase from Alternaria solani

The first natural Diels-Alderase, solanapyrone synthase, was purified 1,630-fold from a crude extract. The 41-kDa protein on SDS-polyacrylamide gel electrophoresis was identified as truncated solanapyrone synthase, and its N-terminal amino acid sequence was found to be QETQNLNNFLESNAINP.     

Purification and N-terminal amino acid sequence determination of the cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei.

The cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151 was purified to homogeneity by anion-exchange and hydrophobic-interaction chromatography, chromatofocusing and gel-filtration. The purification resulted in a 600-700-fold increase in specific activity of the proteinase and the final yield was approximately 20%. Upon chromatofocusing, two proteolytically active components, termed pro135 and pro110, were detected. pro135 had an isoelectric point of 4.2. It had an Mr of about 300,000 as determined by gel-filtration and 135,000 as judged by SDS-PAGE, indicating that it may exist as a dimer in its native state. pro110 had an isoelectric point of 4.4, and an Mr of about 150,000 as determined by gel-filtration and 110,000 as judged by SDS-PAGE. pro110 appears to be a degradation product of pro135 as they have the same N-terminal amino acid sequence. The first N-terminal amino acid was ambiguous for both components, whereas the sequence from the second to the ninth amino acid was Ala-Lys-Ala-Asn-Ser-Met-Ala-Asn. This is identical to the corresponding sequence of the lactococcal cell-wall-bound proteinases. Although the Lactobacillus proteinase was a little smaller than the lactococcal proteinase, their purification characteristics were very similar, suggesting that these proteinases are related.     

Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties

The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Purification and properties of acid phosphatases isolated from Owenia fusiformis.

Acid phosphatase (EC. 3.1.3.2) has been separated by molecular sieving into two fractions and these fractions were purified by Sephadex ion-exchange chromatography. One of the purified enzymes (fraction II) was purified 830 fold and had a specific activity of 34 international units per mg protein at 37 degrees C and at a pH of 4.9. The Km value with p-nitrophenylphosphate as substrate was 9.10(-4) M and the kinetic studies showed no possibilities of control by allosteric transitions, and no effect of metabolites (amino acids) on the reaction velocity.