y-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all -y-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and y-glutamyl-p-nitroanilide as y-glutamyl donors in the transpeptidation reaction were both 35 ,uM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.

The DNA sequence of ggt, the gene that codes for -y-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12, has been determined. The sequence contains a single open reading frame encoding the signal peptide and large and small subunits, in that order. This result suggests that E. coli y-glutamyltranspeptidase is processed posttranslationally.-y-Glutamyltranspeptidase (GGT) ( (29), but the mechanism of its regulation remains to be elucidated.GGT consists of one large and one small subunit (18,28,34). Mammalian GGTs are synthesized as single propolypeptides and then processed into large and small subunits (9,16,19,20). It was of interest to us to determine whether E. coli GGT is also synthesized as a single polypeptide and processed later.To obtain information on these points, we performed DNA sequencing of E. coli K-12 ggt in this study.The nucleotide sequence of E. coli DNA inserted in pSH101 (27) was determined in both directions by the M13 dideoxy-sequencing method (23,26,35) Fig. 1. The large subunit is located proximal to the N terminus and the small subunit is distal to it. We looked upstream for the possible initiation codon, Shine-Dalgarno sequence (25), and promoter; the likeliest ones are shown in Fig. 1 (2,3,9,16,19,20,32). This kind of processing is very rare in procaryotes, but some examples have been reported (21,24,31). In the case of penicillin acylase, a spacer sequence between two subunits is known (21, 24), but that kind of sequence was not reported in E. coli S-adenosylmethionine decarboxylase (31) and mammalian GGTs (5,10,15,17,22). Since the molecular weight of the large subunit of E. coli GGT was 39,200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (28), which coincides with the molecular weight calculated from the sequence data (39197.68), the proteolytic cleavage is thought to occur only between Gln-390 and Thr-391 and between Ala-25 and Ala-26.The secondary structure at this site (Gln-390 and Thr-391) was calculated by the method of Chou and Fasman (4) to be a turn, which is preferentially recognized by signal peptidases (8). However, since signal peptidase II shows preferential cleavage between glycine and cysteine and since signal peptidase I prefers the C terminus of a signal peptide to be an amino acid with a small side chain, such as alanine, serine, or glycine (8), neither of these signal peptidases is likely to be the protease responsible for the cleavage between Gln-390 and Thr-391. More studies are needed to determine when, where, and how the processing into large and small subunits takes place and what kind of protease is responsible for this processing.A likely signal peptide consisting of 25 amino acids was found at the N terminus, with the cleavage occurring between No DNA sequence showing high similarity with the consensus sequence for the u70 promoter was found at the N terminus. Since E. coli GGT is best expressed at 20°C and is expressed only very weakly at 37°C (29), it might be one of the cold shock proteins (13). In E. coli, the heat sh...

Escherichia coli cells showed maximum activity of y-glutomyltranspeptidase (EC 2.3.2.2) when they were grown at 20°C, 14% of maximum activity at 37°C, and none at 43°C. The enzyme activity of intact cells grown at 20°C was stably maintained after the temperature was changed to 45°C. The activity increased during the exponential phase, and maximum activity was found at stationary phase. Its intracellular localization in the periplasmic space was confirmed.-y-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of -y-glutamyl compounds (hydrolyase activity glutaminase activity) and the transfer of their -y-glutamyl moieties to other amino acids and peptides (transferase activity) (24). Although a number of studies on GGT of mammalian cells have been performed and reviewed (1,32,33), the physiological roles of the enzyme are still controversial. GGT of yeasts was also investigated (15,26).Since Talalay (31) found GGT activity in Proteus vulgaris, several studies have been performed on GGT of bacteria, including an L-glutamate-fermenting bacterium (6) and Bacillus natto (2). Nakayama et al. in our laboratory reported that P. mirabilis exhibits high GGT activity (17), and then they purified and characterized it (18) and determined its localization in the cell (19). They also synthesized yglutamyl-L-3,4-dihydroxyphenylalanine (DOPA) enzymatically, using the GGT from P. mirabilis (16), and Togari et al. suggested that this y-glutamyl-L-DOPA is useful as a precursor of L-DOPA in rat brain (manuscript in preparation). Kiuchi et al. (9) also showed that this -y-glutamyl-L-DQPA is useful as a substrate for measuring the enzymatic activity of GGT.Milbauer and Grossowicz (12), Szewczuk and Mulczyk (29), and Nakayama et al. (20) found GGT activity in various bacteria, but they also found that Escherichia coli, the best-studied bacteria in laboratories, does not exhibit strong GGT activity.In this study we found that the GGT activity of E. coli K-12 is greatly affected by growth temperature and growth phase. The localization of this enzyme was also determined by cell fractionation involving osmotic shock or lysozyme treatment. MATERIALS AND METHODSReagents. y-Glutamyl-p-nitroanilide, glycylglycine, and bovine serum albumin were purchased from Wako Pure Chemical Co., lysozyme, o-nitrophenyl-,-D-galactopyranoside, and 5'-AMP were from Sigma Che,mical Co., tryptone was from Difco Laboratories, and yeast extract was from the Oriental Yeast Co. Other chemicals were the best reagents grade available from commercial sources.Bacterial strains and cultures. P. mirabilis was the same strain that Nakayama et al. described (18). Prototrophic E. coli K-12 MG1655 (4) was donated by Carol A. Gross. * Corresponding author. LB broth (13) was used unless otherwise stated. A temperature gradient incubator, model TN-3 (Toyo Kagaku Sangyo Co.), was used in an air-conditioned room at 28°C to create temperature gradients for cultures. In other cases, cells were grown in a 20°C water bath with reciprocal shaking. Precultures were grown at 37°C unle...

The human T‐lymphotropic virus type I (HTLV‐I) is etiologically linked to adult T‐cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV‐I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV‐I in rabbits and had a protective effect against HTLV‐I infection.

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N-P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.