Cloning of cDNA encoding a new peptide C-terminal alpha-amidating enzyme having a putative membrane-spanning domain from Xenopus laevis skin

A cDNA clone encoding a precursor of a peptide C-terminal alpha-amidating enzyme (AE-I) from Xenopus laevis skin was recently isolated and sequenced in our laboratory. In this study, by using the restriction fragment of this clone as a hybridization probe, we have identified the cDNA encoding another new peptide C-terminal alpha-amidating enzyme (tentatively named AE-II) distinct from AE-I. The cDNA encodes a polypeptide of 875 amino acid residues, which contains a region extensively homologous to AE-I precursor at N-terminus. The encoded protein characteristically has a putative membrane-spanning domain near C-terminus. Our results indicate that C-terminal alpha-amide formation of peptides in Xenopus skin is regulated by at least two distinct alpha-amidating enzymes.     

Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase.

The amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (BDH), a phosphatidyl-choline-dependent enzyme, has been determined for the enzyme from rat liver by a combination of nucleotide sequencing of cDNA clones and amino acid sequencing of the purified protein. This represents the first report of the primary structure of this enzyme. The largest clone contained 1435 base pairs and encoded the entire amino acid sequence of mature BDH and the leader peptide of precursor BDH. Hybridization of poly(A+) rat liver mRNA revealed two bands with estimated sizes of 3.2 and 1.7 kb. A computer-based comparison of the amino acid sequence of BDH with other reported sequences reveals a homology with the superfamily of short-chain alcohol dehydrogenases, which are distinct from the classical zinc-dependent alcohol dehydrogenases. This protein family, initially discerned from Drosophila alcohol dehydrogenase and bacterial ribitol dehydrogenase, is now known to include at least 20 enzymes catalyzing oxidations of distinct substrates.     

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Peptide alignment of the porcine 21-hydroxylase cytochrome P-450 using a cDNA sequence of the corresponding bovine enzyme

The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established.     

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.     

An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin

We have purified to homogeneity the enzyme in the kidney cortex which accounts for the vast majority of matrix-degrading activity at neutral pH. The purified enzyme has an apparent molecular mass of 350 kD by gel filtration and of 85 kD on SDS-PAGE under reducing conditions; and it degrades laminin, type IV collagen and fibronectin. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by other proteinase inhibitors. The enzyme was not activated by organomercurials or by trypsin and was not inhibited by tissue inhibitors of metalloproteinases indicating that it is distinct from the other matrix- degrading metalloproteinases. Unexpectedly, the amino acid sequence of the NH2-terminal and two internal peptides of the enzyme showed complete homology to those alpha subunits of rat meprin, an enzyme previously shown to degrade azocasein and insulin B chain but not known to degrade extracellular matrix components. Immunoprecipitation studies, Western blot analyses and other biochemical properties of the purified enzyme confirm that the distinct matrix-degrading enzyme is indeed meprin. Our data also demonstrate that meprin is the major enzyme in the renal cortex capable of degrading components of the extracellular matrix. The demonstration of this hitherto unknown function of meprin suggests its potential role in renal pathophysiology.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.     

Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

Beta subunit of rat liver mitochondrial ATP synthase: cDNA cloning, amino acid sequence, expression in Escherichia coli, and structural relationship to adenylate kinase

The amino acid sequence of all but a few N-terminal residues of the beta subunit of rat liver ATP synthase has been determined from cDNA clones. Rat liver F1-beta is shown to contain 17 amino acid differences from that reported for F1-beta of bovine heart, 2 differences of which involve differences in charge. This may account in part for the observation that bovine heart F1 binds nucleotides with much greater affinity than the rat liver enzyme. Rat liver F1-beta also contains homologous regions with another nucleotide binding protein, adenylate kinase, for which high-resolution structural studies are available. Adjacent to one of these homologous regions is an eight amino acid stretch which bears striking homology to the phosphorylation region of the (Na+,K+)-ATPase. The combination of these two homology regions may constitute at least part of a nucleotide binding domain in F1-beta. Significantly, both rat liver and bovine heart beta contain these regions of homology, whereas the 17 amino acid differences between the two enzymes lie outside this region. The possibility of a second nucleotide binding domain which differs between the two enzymes is discussed. A cDNA clone containing all the regions of homology as well as 11 of the 17 amino acid differences between the bovine heart and rat liver beta subunits has been ligated into the bacterial expression vector pKK223-3. After transformation of a protease-deficient strain of Escherichia coli, this cDNA clone is expressed as a 36-kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver UDP-glucuronosyltransferase. cDNA sequence and expression of a form glucuronidating 3-hydroxyandrogens

A cDNA clone, pUDPGTr-4, encoding a form of rat UDP-glucuronosyltransferase has been isolated from a SV40 expression library. Sequence analysis revealed that the cDNA is 1970 base pairs in length and encodes a protein of 530 amino acids, which has amino- and carboxyl-terminal sequences characteristic of signal peptide and transmembrane segments, respectively. There is one potential asparagine-linked glycosylation site. Transfection of UDPGTr-4 cDNA into COS cells resulted in the glucuronidation of etiocholanolone, androsterone, and lithocholic acid in a transient expression assay. Several other common substrates of UDP-glucuronosyltransferase were not conjugated by the UDPGTr-4 enzyme. UDPGTr-4 cDNA is identical in sequence over a common 1.7 kilobase-region of overlap to UDPGTr-1, a cDNA previously isolated in this laboratory (Mackenzie, P. I., Gonzalez, F. J., and Owens, I. S. (1984) J. Biol. Chem. 259, 12153-12160). UDPGTr-4 cDNA, however, contains a shorter 3'-untranslated region. Northern analysis showed that the poly(A) RNA counterparts of UDPGTr-4 and UDPGTr-1 cDNAs are approximately 2.3 and 3.0 kilobases in length, respectively. The steady-state level of UDPGTr-4 poly(A) RNA in the liver is 20-fold higher than that of UDPGTr-1 poly(A) RNA. These data indicate that the UDPGTr-4 enzyme is a 3-hydroxyandrogen UDP-glucuronosyltransferase which is encoded by two distinct species of mRNA transcribed from the same gene.     

Cloning and sequence analysis of a cDNA for human glycosylasparaginase. A single gene encodes the subunits of this lysosomal amidase

We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the alpha and beta subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (alpha) and 15 (beta) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the alpha and beta subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.     

Isolation of cDNA clones coding for rat isovaleryl-CoA dehydrogenase and assignment of the gene to human chromosome 15

Rat liver mRNA encoding the cytoplasmic precursor of mitochondrial isovaleryl-CoA dehydrogenase was highly enriched by polysome immunopurification using a polyclonal monospecific antibody. The purified mRNA was used to prepare a plasmid cDNA library which was screened with two oligonucleotide mixtures encoding two peptides in the amino-terminal portion of mature rat isovaleryl-CoA dehydrogenase. Thirty-one overlapping cDNA clones, spanning a region of 2.1 kbp, were isolated and characterized. The cDNA sequence of a 5'-end clone, rIVD-13 (155 bp), predicts a mitochondrial leader peptide of 30 amino acid residues and the first 18 amino acids of the mature protein. These consecutive 18 residues completely matched the amino-terminal peptide determined by automated Edman degradation of the rat enzyme. The leader peptide contains six arginines, has no acidic residues, and is particularly rich in leucine, alanine, and proline residues. Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated rat cDNA (2 kbp) assigned the isovaleryl-CoA dehydrogenase gene to the long arm of chromosome 15, region q14----qter. The chromosomal assignment was confirmed and further refined to bands q14----q15 by in situ hybridization of the probe to human metaphase cells. This location differs from that of the gene for medium-chain acyl-CoA dehydrogenase, a closely related enzyme, which has been previously assigned to chromosome 1.     

Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A) + RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.     

The sequence of human parathymosin deduced from a cloned human kidney cDNA

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine. Analysis of the 5' flanking sequence excluded the presence of a hydrophobic signal peptide in the translated sequence. It may therefore be concluded that parathymosin, like prothymosin alpha, is synthesized without formation of a large precursor polypeptide. Comparison of the deduced amino acid sequence with the known primary structure of rat and bovine parathymosins shows that the primary structure of parathymosin is highly conserved among these species.     

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.