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We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase. Similar studies with Escherichia coli ribosomes, using antibodies directed against the enzyme beta-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, approximately 75 A from the interface between subunits and nearly 160 A from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.

We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase. The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm. One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined. The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20. The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins.

The site of attachment to beta-galactosidase of the active site-directed inhibitor, beta-D-galactopyranosylmethyl p-nitrophenyl triazene, was determined. When the enzyme is completely inactivated, 1 mol of the galactopyranosylmethyl group is bound per mol of monomer with retention of the tetrameric structure. After reaction with the [14C]methyl reagent, labeled peptides were isolated and analyzed. The radioactive label was found to be covalently bound to methionine residue 500.

A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.

alpha Complementation in beta-galactosidase is the restoration of enzyme activity by addition of the alpha donor CNBr2, from amino acid residues 3--92 of the polypeptide, to inactive M15 protein from the lacZ deletion mutant strain M15. M15 protein lacks residues 11--41 and is a dimer; the active complex, like native beta-galactosidase, is tetrameric [Langley, K. E., & Zabin, I. (1976) Biochemistry 15, 4866--4875]. A dimer--dimer binding region in beta-galactosidase has been identified by proteolytic and immunologic studies of alpha-complementation. Proteolytic experiments were carried out with trypsin. Treatment of native beta-galactosidase with trypsin, followed by reaction of the mixture with cyanogen bromide, yields intact CNBr2 as measured by its ability to complement M15 protein. Active CNBr2 is not obtained when urea-denatured beta-galactosidase is treated in the same way. Therefore the segment corresponding to CNBr2 is apparently buried within the folded protein. Immunologic experiments were carried out with antibodies against CNBr2, tryptic peptide T8 (residues 60--140), and CNBr3 (residues 93--187). Anti-CNBr2 and anti-T8 bind to M15 protein but not to beta-galactosidase, indicating that this area is exposed in the dimer. Anti CNBr2, but not anti-T8 or anti-CNBr3, inhibits the formation of alpha-complemented enzyme. These results indicate that an early part of the sequence, within the segment corresponding to CNBr2, is involved in dimer--dimer interaction.

Beta-Galactosidase is rapidly inactivated by iodination catalyzed by lactoperoxidase but is not inactivated in the presence of the substrate analogue, isopropyl beta-D-thiogalactoside (IPTG). Enzyme activity is lost upon the incorporation of 1 mol of iodine per mol of monomer, without dissociation of the tetrameric structure. Tryptic digests of beta-galactosidase iodinated with 125I in the presence and absence of IPTG were separated by high-performance liquid chromatography and were compared. One fraction was found to be more highly labeled in the digest from the inactivated protein. After isolation of the peptide, amino acid analysis indicated it to be Asp-Tyr-Leu-Arg, residues 252-255. Thus, Tyr-253 is the most reactive tyrosine in beta-galactosidase. This suggests that the conformation of this region of the protein may be altered by binding of IPTG to make Tyr-253 less accessible to iodination. Alternatively, Tyr-253 could be an active-site residue.

The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell by virtue of an amino-terminal signal sequence. Using DNA sequencing, we have determined the precise nature of mutations in the signal sequence which prevent the export of the maltose binding protein, causing it to accumulate in the cytoplasm in its precursor form. In most cases, the change of a single hydrophobic or uncharged amino acid to a charged amino acid within the signal sequence is sufficient to block the secretion process.

In previous studies, a cyanogen bromide peptide derived from amino-acid residues 3-92 of beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) was shown to have alpha-donor activity in intracistronic alpha-complementation. We have now isolated the defective beta-galactosidase alpha-acceptor protein from the deletion mutant strain M15 of Escherichia coli and find that it lacks residues 11-41 of betal-galactosidase. This is demonstrated by the isolation and sequence determination of a cyanogen bromide peptide from the M15 protein, which is identical to the corresponding peptide from beta-galactosidase except for the missing amino acids. We conclude that the alpha-donor peptide restores the region missing in the M15 protein.

Extracts of thymic lymphoma that are obtained from AKR mice and are kept in the cold for at least several days can induce lipolytic activity in rat adipocyte suspensions. Freshly prepared extracts have low activity but contain a low molecular weight material of less than 10,000 daltons that aggregates on standing in the cold and becomes active. Treatment of aged extracts with trypsin causes a loss in activity indicating that the active material is a protein. It has been obtained in partially purified form, is relatively heat stable, and is not a lipase. Activity was also demonstrated in AKRXDBA/2 lymphoma (induced by AKR SL3-3 virus) and in transplanted lymphomas from a Friend-virus-induced erythroleukemia cell line in DBA/2 mice, but was not detected in normal thymus, spleen, liver, or other tissues. The partially purified material produced a massive fat mobilization when injected into normal mice.

The sequence of the COOH-terminal third (omega) of beta-galactosidase is presented. The size of the 7 cyanogen bromide peptides of this segment is larger on the average, about 52 amino acid residues as compared to an average size of 42 for cyanogen bromide peptides in the whole molecule. Tyrosine, phenylalanine, and valine are low in this segment whereas alanine and lysine are high. This region has a slight excess of basic groups.

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.

The amino terminal sequence of the maltose-binding protein as well as the leader region were reported earlier [2]. The amino acid composition of the protein indicated that the protein has no cysteine and very few methionine, arginine and histidine residues. In contrast, the residues of lysine, alanine and aspartic acid (or asparagine) account for about a third of the approximately 360 amino acid residues in the protein. Five cyanogen bromide peptides ranging in size from 10 to 154 residues have now been isolated by Sephadex G50 and G100 gel filtration. These five peptides account for the whole protein. Sequence determination of these fragments as well as of others has now been initiated.

Escherichia coli strains have been constructed in which lacZ, the gene for the cytoplasmic enzyme beta-galactosidase, is fused to lamB, the gene for an outer membrane protein. One such strain produces a beta-galactosidase which remains cytoplasmic even though it possesses the complete signal sequence of the lamB protein precursor at the amino-terminal end.

Antibodies were prepared against 18 tryptic and cyanogen bromide peptides from beta-galactosidase ranging in size from 15 to 96 amino acid residues representing more than 80% of the polypeptide chain. They were tested for binding capacity and affinity toward their homologous antigens and toward the whole native protein. Nine antisera bound to beta-galactosidase; these had been raised against certain peptides from the central and carboxyl-terminal regions of the poly-peptide chain. Based on these results a preliminary model of the three-dimensional structure of the folded protein is suggested.

Studies on beta-galactosidase alpha-complementation are reviewed. The isolation and structure of two beta-galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole beta-galactosidase; the other is a dimeric protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. alpha-Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the functionally important segment. The effect on alpha-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for beta-galactosidase structure and for proteins in general are discussed.

The amino acid sequence of beta-galactosidase was determined. The protein contains 1021 amino acid residues in a single polypeptide chain. The subunit molecular weight calculated from the sequence is 116,248. The sequence determination, carried out mainly by conventional methods, was aided by complementation tests, by the use of termination mutant strains, and by a new immunochemical method. The five residue sequence Thr-Pro-His-Pro-Ala appears twice within the polypeptide chain, but no other striking homologous features are evident.

The positions of three Escherichia coli lacZ operator-proximal nonsense mutations and one deletion mutation have been determined. The nonsense mutations were suppressed with supF, resulting in the production of active beta-galactosidase by each strain. Amino acid sequencing identified the positions of the tyrosine residues inserted by supF, and thereby established that nonsense mutations lacZ2, lacZ2246, and lacZU131 are at sites corresponding to amino acids 23, 36, and 41 of beta-galactosidase, respectively. The deletion mutant, lacZM112, produced a dimeric beta-galactosidase protein missing amino acid residues 23 through 31 of the native enzyme.

Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.

The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant. Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues. A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90. This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer.

The Escherichia coli lacZ ochre mutant strain U118 was converted to an amber mutant and suppressed with supF, which inserts tyrosine. Enzymatically active beta-galactosidase was isolated. It contained tyrosine at residue number 17 instead of glutamic acid as in wild type.

beta-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the beta-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg(-)) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg(-) and Deg(+) strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.

The amino acids in 9 cyanogen bromide peptides have been placed in sequence starting from the NH2 terminus. The peptides account for residues 1 to 377 of the whole protein and include the largest (CNBr7, 119 residues) and the smallest (CNBr1, 2 residues) of the cyanogen bromide peptides. This region contains only 3 of the 20 lysine residues in the polypeptide chain. A high proportion of charged groups are present (28 of 66 arginine, 28 of 60 glutamic acid, and 24 of 65 aspartic acid residues).

The amino acid sequence in the 8 cyanogen bromide peptides comprising the central segment of beta-galactosidase is presented. This portion of the molecule, about 27% of the protein, contains over 40% of the lysine and tyrosine residues and has a slight excess of basic amino acids.

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

The sequences of the malE gene and of its mature product, the maltose-binding protein, have been determined and are in good agreement. The malE gene encodes the pre-protein (396 amino acid residues) which yields, upon cleavage of the NH2-terminal extension (26 amino acid residues), the mature maltose-binding protein (370 amino acid residues). The malE mRNA could form stable stem and loop structures, some of which may account for translational pauses observed by Randall et al. (Randall, L., Josefsson, L. G. & Hardy, S. J. S. (1980) Eur. J. Biochem. 107, 375-379). The sequence change due to an in-frame nonpolar deletion of 765 nucleotides in malE is also presented as well as homologies between the maltose-binding protein and other sugar-binding proteins.

The amino acid sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) has been compared to itself and to other proteins. Two segments, each of about 380 amino acids, comprising the first three-fourths of the polypeptide chain, were found to be very similar to each other. It is concluded that they are homologous. The carboxyl-terminal fourth has a high percentage of amino acid identities with dihydrofolate reductase of Escherichia coli, suggesting these sequences also are homologous. A model for the origin of beta-galactosidase is presented. The overall similarity of beta-galactosidase to lac repressor does not appear to be significant.

The amino acid sequence of beta-galactosidase has been determined. The monomer contains 1,021 amino acid residues in a single polypeptide chain and has a molecular weight of 116,349. All 80 tryptic peptides as well as all 24 CNBr peptides have been isolated in pure form. Evidence is presented for the ordering of the CNBr peptides. The sequence determination was aided by analysis of cyanogen bromide peptides obtained from a polypeptide fragment produced by a lacZ termination mutant strain.

Enzyme activity is restored to two defective beta-galactosidase molecules (M15 protein lacking amino acid residues 11-41 and M112 protein lacking residues 23-31) by incubation with CNBr2 (residues 3-92 of beta-galactosidase). M15 and M112 proteins (alpha-acceptors) are dimers. Complemented enzyme, like wild type, has a tetrameric structure. Cleavage of CNBr2 with glutamic acid-specific protease yielded a much smaller alp ha-donor (3-41 peptide) which was also effective in complementation, indicating that the M15 protein can supply all of the residues from 42-92 for the structure of complemented enzyme. Treatment of M112 protein/3-41 peptide complemented enzyme with trypsin under very mild conditions followed by examination of the products demonstrated that the alpha-donor pep]tide supplies the NH2-terminal segment of complemented enzyme. Similar trypsin treatment of M15 protein/CNBr2 indicated that in this complemented enzyme the polypeptide region beyond those residues missing in the alpha-acceptor can be provided by either the alpha-donor or the alpha-acceptor. Both M15 protein and M112 protein are more susceptible to mild tryptic proteolysis than complemented enzyme, indicating a more open structure. Several antipeptide antibodies that react with these two proteins do not react with beta-galactosidase. M112 protein, like M15 protein, can be activated by anti-beta-galactosidase but to a much higher level.

Intracistronic alpha complementation involving Escherichia coli beta-galactosidase occurs between the cyanogen bromide peptide CB2, derived from residues 3-92 of beta-galactosidase (Langley, K.E., Fowler, A.V., and Zabin, I. (1975), J. Biol. Chem. 250, 2587), and the defective beta-galactosidase from the Z-deletion mutant strain M15. The M15 protein, a dimer, lacks residues 11-41 of beta-galactosidase (Langley, K.E., Villarejo, M.R., Fowler, A.V., Zamenhof, P.J., and Zabin, I. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 1254). The complemented enzyme formed from purified components has a molecular weight of 533 000+/-25 000, is therefore tetrameric, and has a probable stoichiometry of 1 CB2:1 M15 monomer. The complemented enzyme has the same Km for substrate as wild type enzyme, but is less stable to heat or urea treatment. The overall equilibrium constant for the complementation reaction is approximately 1-2 X 10(9) M-1. Initial velocity studies indicate saturation kinetics when either component is fixed and limiting, with an apparent Kd of about 10(-6) M. A first-order rate constant of 0.05-0.1 min-1 was estimated. The kinetics favor a model of rapid complex formation, followed by slow conformational change, as the mechanism of activation. Ultraviolet difference spectroscopy indicated an increased absorbance in the 290-300 nm region as a result of the complementation reaction. The kinetics of the increase suggest that two processes, one rapid and the other slower, could be responsible. The temperature dependence of complementation (Ea approximately 24 000 cal) is also consistent with the rate-determining step being a conformational change.

Intracistronic alpha-complementation between a cyanogen bromide digest of beta-galactosidase and an extract of the lac Zminus operator-proximal deletion mutant M15 was used to monitor the purification of a cyanogen bromide peptide (CB2) responsible for the complementation. Key steps in the purification were ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sephadex in the presence of urea, and Sephadex gel filtration. CB2 contains residues 3 to 92 of beta-galactosidase. Its sequence is: Ile-Thr-Asp-Ser-Leu-Ala-Val-Val-Leu-Gln-Arg-Arg-Asp-Trp-Glu-Asn-Pro-Gly-Val-Thr-Gln-Leu-Asn-Arg-Leu-Ala-Ala-His-Pro-Pro-Phe-Ala-Ser-Trp-Arg-Asn-Ser-Glu-Glu-Ala-Arg-Thr-Asp-Arg-Pro-Ser-Gln-Gln-Leu-Arg-Ser-Leu-Asn-Gly-Glu-Trp-Arg-Phe-Ala-Trp-Phe-Pro-Ala-Pro-Glu-Ala-Val-Pro-Glu-Ser-Trp-Leu-Glu-Cys-Asp-Leu-Pro-Glu-Ala-Asp-Thr-Val-Val-Val-Pro-Ser-Asn-Trp-Gln-Met. Thus no more than 1/13 of the beta-galactosidase polypeptide chain, starting 2 residues from the NH2 terminus, is necessary for alpha-complementation with M15 as alpha-acceptor.

Hydrolysis with trypsin of citraconyl-carboxy-methyl-beta-galactosidase was carried out under limiting conditions. No Asp-Arg-X sequences were cleaved and many large peptides were produced. Butanol extraction from dilute acid proved very useful for separating the more hydrophobic fragments. Peptides were purified and sequenced. From this digest and two earlier preparations, all 80 theoretically possible tryptic fragments have been isolated and their structures determined.

Ribosomes carrying nascent chains of beta-galactosidase were prepared by disruption of Escherichia coli in detergent-free buffer of high salt concentration, followed by purification on a discontinuous sucrose gradient. Assay by the method of immune hemolysis inhibition with anti-beta-galactosidase indicated that considerable amounts of antibody were bound by the growing chains. Much of the crossreacting material could be released from the ribosomes by treatment with puromycin. The ability to bind anti-beta-galactosidase was completely destroyed when ribosomes were heated at 60 degrees C. At very early times after induction, well before the appearance of active enzyme, crossreacting material could be demonstrated on ribosomes; this finding correlated with the appearance of an amino-terminal fragment of beta-galactosidase. Thus, growing chains of beta-galactosidase must begin to fold before their release from the ribosome.