Cloning and characterization of a murine band 3-related cDNA from kidney and from a lymphoid cell line

Anion exchange is a nearly ubiquitous cellular transport function which contributes to the regulation of cell pH and of cell volume. However, the only plasma membrane anion exchanger of known identity and sequence is erythroid band 3. Both hybridization and immunologic data support the presence of band 3-related mRNAs and proteins in nonerythroid tissues. We have used low stringency hybridization with the murine band 3 cDNA to clone a band 3-related cDNA from murine kidney and from 70Z/3 pre-B cells. The cDNA encodes a band 3-related protein (B3RP) of 1237 amino acids, with a predicted mass of 137 kDa. The carboxyl-terminal hydrophobic domain of B3RP has an amino acid sequence 67% identical to that of band 3, with a very similar predicted secondary structure. The amino-terminal hydrophilic domain of B3RP has two sections. The section adjacent to the putative membrane-associated segment is 33% identical in amino acid sequence to the amino-terminal, cytoplasmic domain of band 3. The other, far amino-terminal section of B3RP has no correspondent in the band 3 sequence. B3RP mRNA is present in a variety of epithelial and other tissues and probably encodes an anion exchange protein of wide distribution.     

Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy), playing an essential role in modulating the cellular Hcy levels and regulating activities of a host of methyltransferases in eukaryotic cells. This enzyme exists in an open conformation (active site unoccupied) and a closed conformation (active site occupied with substrate or inhibitor) [Turner, M. A., Yang, X., Yin, D., Kuczera, K., Borchardt, R. T., and Howell, P. L. (2000) Cell Biochem. Biophys. 33, 101-125]. To investigate the binding of natural substrates during catalysis, the computational docking program AutoDock (with confirming calculations using CHARMM) was used to predict the binding modes of various substrates or inhibitors with the closed and open forms of AdoHcy hydrolase. The results have revealed that the interaction between a substrate and the open form of the enzyme is nonspecific, whereas the binding of the substrate in the closed form is highly specific with the adenine moiety of a substrate as the main recognition factor. Residues Thr57, Glu59, Glu156, Gln181, Lys186, Asp190, Met351, and His35 are involved in substrate binding, which is consistent with the crystal structure. His55 in the docked model appears to participate in the elimination of water from Ado through the interaction with the 5'-OH group of Ado. In the same reaction, Asp131 removes a proton from the 4' position of the substrate after the oxidation-reduction reaction in the enzyme. To identify the residues that bind the Hcy moiety, AdoHcy was docked to the closed form of AdoHcy hydrolase. The Hcy tail is predicted to interact with His55, Cys79, Asn80, Asp131, Asp134, and Leu344 in a strained conformation, which may lower the reaction barrier and enhance the catalysis rate.     

Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Polymorphism of S-adenosylhomocysteine hydrolase in Italy

S-adenosylhomocysteine hydrolase (SAHH) polymorphism has been investigated in the Italian population. Three common alleles, SAHH*1, SAHH*2 and SAHH*3, have been observed and the estimated gene frequencies are 0.968, 0.023 and 0.009, respectively. SAHH activity has been assayed in 50 healthy individuals and the mean activity was 0.043 +/- 0.017 mumol uric acid/min/g Hb at 37 degrees C. Five heterozygotes for adenosine deaminase deficiency and three heterozygotes for purine nucleoside phosphorylase deficiency showed SAHH within the range of the normal distribution. The effects of some thiol reagents on red blood cell SAHH electrophoretic pattern have been investigated.     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

Selection and characterization of a carrot cell line tolerant to glyphosate

Cultured carrot (Daucus carota L.) cells were adapted to growing in 25 millimolar glyphosate by transfer into progressively higher concentrations of the herbicide. Tolerance was increased 52-fold, and the adaptation was stable in the absence of glyphosate. The uptake of glyphosate was similar for adapted and nonadapted cells. Activity of the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase was 12-fold higher in the adapted line compared to nonadapted cells, while activities of shikimate dehydrogenase and anthranilate synthase were similar in the two cell types. The adapted cells had higher levels of free amino acids—especially threonine, methionine, tyrosine, phenylalanine, tryptophan, histidine, and arginine—than did nonadapted cells. Glyphosate treatment caused decreases of 50 to 65% in the levels of serine, glycine, methionine, tyrosine, phenylalanine, and tryptophan in nonadapted cells, but caused little change in free amino acid levels in adapted cells.

The adaptation reported here supports the growing body of evidence linking tolerance to glyphosate with increased levels of the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The elevated levels of aromatic amino acids, which may confer resistance in adapted cells, suggest that control of the shikimate pathway may be altered in these cells.

     

Biochemical genetic analysis of the role of methylthioadenosine phosphorylase in a murine lymphoid cell line

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.     

Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.     

Establishment and characterization of a malignant lymphoid cell line from a chronic lymphocytic leukemia patient

A long-term culture Epstein-Barr virus (EBV)-negative malignant lymphoid cell line (NAK) was established from a lymph node biopsy of a chronic lymphocytic leukemia patient. This cell line is of particular interest because it grows as an adherent cell line and depends on the presence of autologous conditioned medium for growth. After 6 months of growth in vitro, doubling time and cell cycle parameters were derived. Doubling time was 48 hours with over 45% cycling cells. Cell viability was over 90%. Expression of B-cell markers (CD19 and CD20) and surface immunoglobulin of the original tumor cell biopsy were roughly the same as in passage 14 (3 months in culture), including the expression of the original patient idiotype and IgM-lambda. Furthermore, binding of antiidiotypic antibodies was only slightly decreased at passage 14. Cytogenetic studies of chromosomal abnormalities in the primary tumor tissue and in later passages indicated similar abnormalities, with no translocations t(8;14), t(14;22), or t(2;8). However, frequent trisomies, deletions, and t(1;4) translocations were observed. Negative results for EBV nuclear antigen indicate that this cell line is an EBV-negative cell line.     

Purification and characterization of a collagenous protein secreted by a murine teratocarcinoma-derived cell line

A collagenous protein could be precipitated by (NH₄)₂SO₄ from the culture medium of a murine teratocarcinoma-derived cell line (Ko, C.Y., Johnson, L.D. and Priest, R.E. (1979) Biochim. Biophys. Acta 581, 252–259). Further purification of this protein was achieved by combining DEAE-cellulose chromatography with either CM-cellulose or molecular sieve chromatography. The collagenous polypeptides had subunit molecular weights of 160 000, if determined by molecular sieve chromatography, or 190 000, if determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they are not linked by disulphide bridges. Amino acid composition of this collagen is similar to that of a murine type IV collagen isolated from EHS sarcoma (Timpl et al. (1978) Eur. J. Biochem. 84, 43–52). The most prominent peptides resulting from cleavage of the protein by CNBr had estimated molecular weights of 25 000, 23 000, 11 700 and 9400. Pepsin treatment of this collagen under non-denaturing conditions produced three major fragments having molecular weights of 70 000, 45 000 and 43 000. We conclude that the collagen secreted by the murine teratocarcinoma-derived cell culture is a type IV basement membrane collagen. Therefore, this culture system should provide a continuous source of type IV collagen, which may be used to study the interaction of this collagen with other basement membrane components.     

Isolation and characterization of cell cycle-enriched subpopulations of a murine macrophage cell line by centrifugal elutriation

The cell cycle of the P388D 1 murine macrophage line was delineated and suspensions of exponentially growing cells were separated by centrifugal elutriation into subpopulations enriched in the various phases of the cycle. Analysis of both growth and labelled mitoses curves disclosed that the doubling and cell-cycle times were essentially identical (18.4 and 18.3 h), indicating that all cells were in cycle. In addition, G1 + 1/2M was 4.3 h, whereas S phase and G2 + 1/2M lasted about 12 and 1.5 h. The most homogeneous subpopulations of phase-enriched cells obtained by elutriation were cells in G1 and S, where purities (estimated by both labelling indices and analyses of DNA histograms obtained by flow cytometry) exceeded 80%. Isolation of G2 + M-phase cells was not as efficient, although the purity of these subpopulations was consistently greater than of 50%, an approx. 10-fold enrichment over unseparated suspensions of cells. Comparison of IgG2a-Fc-receptor-mediated phagocytic activities among the phase-enriched subpopulations showed that cells in G2 had appreciably enhanced activity.     

S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum: cloning, overexpression, purification, and biochemical characterization

The S-adenosylhomocysteine hydrolase gene (sahase) was cloned from the Gram-positive soil bacterium Corynebacterium glutamicum (ATCC 13032) and sequenced. The sahase gene possesses an open reading frame, which consists of 1,434 nucleotides that encode 478 amino acids. The sahase gene from C. glutamicum was expressed in Escherichia coli Rosetta cells by inserting the 1,434-bp fragment downstream from the isopropyl-beta-D-thiogalactopyranoside-inducible promoter of the pET28a+ expression vector. The recombinant S-adenosylhomocysteine hydrolase from C. glutamicum (CgrSAHase) was purified efficiently by a two-step procedure, tangential ultrafiltration and affinity chromatography. The molecular weight of the CgrSAHase, estimated by gel filtration, was about 210 kDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 52 +/- 1 kDa. The Michaelis-Menten constants for the natural substrates of the enzyme, S-adenosylhomocysteine (SAH), adenosine, and homocysteine, were determined to be 12, 1.4, and 40 microM, respectively. The overexpression of CgrSAHase was achieved at high level (>40 mg protein/g wet cells). Because of its high capacity to synthesize SAH, this enzyme is of high biotechnological interest.     

Localization of S-adenosylhomocysteine hydrolase in the rat kidney.

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 +/- 0.02 mU/mg in the kidney, 0.32 +/- 0.03 mU/mg in the glomeruli, and 0.50 +/- 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation. (J Histochem Cytochem 48:211-218, 2000)     

Gene frequencies of S-adenosylhomocysteine hydrolase (SAHH) in a Japanese population

Summary Genetic polymorphism of S-adenosylhomocysteine hydrolase (SAHH) was investigated in a total of 214 red blood cell samples from unrelated Japanese using the starch gel electrophoresis and the enzyme-specific staining procedures. Three common phenotypes were observed which corresponded to SAHH 1, SAHH 2-1, and SAHH 2, controlled by two alleles, SAHH^*1 and SAHH^*2. The estimated gene frequencies of SAHH^*1 and SAHH^*2 in Japanese were 0.953 and 0.047, respectively. This result was not different from European samples reported by Bissbort et al. (1983).     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).