Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.     

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.     

Molecular evolution of urea amidolyase and urea carboxylase in fungi

Background  

Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms.     

Subunit structure of Escherichia coli exonuclease VII

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes.     

Cloning and nucleotide sequence of the urea amidolyase gene from Candida utilis

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida utilis CA(u)-37 (DUR1,2^c) was cloned by plaque hybridization, and the nucleotide sequences of DUR1, 2^c and its flanking regions were determined. DUR1, 2^c was found to be composed of 5,490 base pairs and 1,830 amino acid residues. Using Edman degradation of the purified enzyme, it was revealed that the amino-terminal residue (methionine) was processed for maturation. A TATA-box like sequence was found 112 bases upstream from the translation start site (ATG). The site of the poly (A) tail was found 54 bases downstream from the translation stop site (TGA), since cDNA of DUR1, 2^c was synthesized from mRNA and sequenced. The nucleotide sequences of the urea amidolyase gene from Saccharomyces cerevisiae and DUR1, 2^c were very similar to each other (65.3%), as were the deduced amino acid sequences (67.2%). The molecular weight of DUR1, 2^c was calculated to be 200,700. This value corresponded to the result obtained from SDS-polyacrylamide gel electrophoresis of the purified enzyme. The enzyme functions in a dimeric form. Three important regions were found in the amino acid sequence of urea amidolyase through the homology search. It was predicted that each region was equivalent to the active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of the DUR1, 2^c gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.     

Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) was purified from Streptomyces clavuligerus by a combination of salt precipitation, ultrafiltration, and anion-exchange chromatography. The final purified material gave two protein bands with molecular weights of 283,000 and 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis in nondenaturing gels gave a single protein band with an estimated molecular weight of 560,000. These results suggest that ACVS is a multimer composed of nonidentical subunits.     

A multifunctional calmodulin-stimulated phosphatase

This review summarizes current knowledge concerning structure-function, substrate specificity, localization, and regulatory properties of calcineurin. Calcineurin is composed of two nonidentical subunits, one of which is responsible for catalytic activity and calmodulin binding while the other subunit contains four high-affinity Ca2+-binding sites. The enzyme possesses calmodulin-stimulated and metal ion-dependent phosphatase activity toward several nonprotein and phosphoseryl-, phosphothreonyl- and phosphotyrosyl-containing protein substrates. These recent results suggest that the protein may play a multifunctional role in interactions between the Ca2+/CaM second messenger system and other second messenger systems.     

Hominoid triosephosphate isomerase: Characterization of the major cell proliferation specific isozyme

Summary Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35^S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [³⁵S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains an blocked NH₂-terminus and length heterogenity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.     

Urease and urea amidolyase: Determination of activity in liverworts

Using highly sensitive techniques, we have investigated urea degradation in the liverworts and found that they have high urease but no detectable urea amidolyase activity.     

Subunit structure of electron transfer flavoprotein

The electron transfer flavoprotein from pig liver mitochondria is a 57,000-dalton electron transferase which links several primary flavoprotein dehydrogenases with the mitochondrial electron transport system. The protein was previously reported to be a dimer of apparently identical subunits. There are conflicting estimates in the literature regarding the FAD content of the protein. The results presented here clearly show that the protein contains nonidentical subunits based on polyacrylamide gel electrophoresis in the presence of 8 M urea and sodium dodecyl sulfate. The molecular weights of the subunits are 31,000 and 27,000. Analysis of peptides generated by cleavage of the subunits with cyanogen bromide show that the subunits have different primary structures. This result and amino acid analyses of the protein and the purified subunits show that the heterogeneity cannot be due to proteolysis. Using an experimentally determined molar extinction coefficient for the protein-bound flavin, a minimum Mr = 55,000 was calculated, indicating that the protein contains 1 mol of FAD/mol of protein.     

Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.     

Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.     

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus.

The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.     

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.     

Mouse DNA polymerase alpha. Subunit structure and identification of a species with associated exonuclease.

Two species of alpha-polymerase with very similar catalytic properties have been purified to near homogeneity from a soluble protein fraction of mouse myeloma. Sedimentation analysis in 0.5 M salt-containing glycerol gradients indicated that both species had a native Mr of about 190,000. Each species contained nonidentical subunits with apparent molecular weights of about 47,000 and 54,000. Subunits of Mr = approximately 50,000 had been found previously in calf thymus alpha-polymerase (Holmes, A. M., Hesslewood, I. P., and Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499; (1976) Eur. J. Biochem. 62, 229-235). Tryptic peptide mapping failed to reveal primary structure homology between the subunits of the two enzymes. Thus, the two alpha-polymerases are clearly different species. These two enzymes are further distinguished by the fact that one of them has associated exonuclease activities. One activity degraded single-stranded DNA to mononucleotides in the 3' leads to 5' direction and acted distributively. The other exonuclease activity also degraded single-stranded DNA to mononucleotides, but this degradation was in the 5' leads to 3' direction in a processive fashion. Both exonuclease activities co-migrated with the polymerase activity during the final purification step of polyacrylamide gradient gel electrophoresis, which yielded the essentially homogenous alpha-polymerase, and also during sedimentation of the purified enzyme through a high salt glycerol gradient.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

Rapid Regulation of an Anthranilate Synthase Aggregate by Hysteresis

The anthranilate synthase aggregate from Bacillus subtilis is composed of two nonidentical subunits, denoted E and X, which are readily associated or dissociated. A complex of subunit E and X can utilize glutamine or ammonia as substrates in the formation of anthranilate. Partially purified subunit E is capable of using only ammonia as the amide donor in the anthranilate synthase reaction. The stability of the EX complex is strongly influenced by glutamine and by the concentrations of the subunits. Glutamine stabilizes the aggregate as a molecular species in which the velocity of the glutamine-reactive anthranilate synthase is a linear function of protein concentration. In the absence of glutamine the aggregate is readily dissociated following dilution of the extract; that is, velocity concaves upward as a function of increasing protein concentration. Reassociation of the EX complex is characterized by a velocity lag (or hysteretic response) before steady-state velocity for the glutamine-reactive anthranilate synthase is reached. We propose that association and dissociation of the anthranilate synthase aggregate may be physiologically significant and provide a control mechanism whereby repression or derepression causes disproportionate losses or gains in activity by virtue of protein-protein interactions between subunits E and X.     

Purification of bovine kidney and heart pyruvate dehydrogenase phosphatase on Sepharose derivatized with the pyruvate dehydrogenase complex

Pyruvate dehydrogenase phosphatase has been purified to apparent homogeneity from mitochondrial extracts of both beef heart and beef kidney. An essential step in this three-step purification is affinity chromatography of a largely purified phosphatase fraction using Sepharose beads to which pyruvate dehydrogenase complex is covalently bound through the lipoic acid residues of the dihydrolipoyl transacetylase component of the complex. The purified phosphatase, which has a native relative molecular mass, Mr, of about 140000, is composed of two nonidentical subunits of Mr 89000 and 49000.     

The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.