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We have determined the chromosomal location of the human gene for gamma-glutamyltransferase (GGT). This study was done by in situ hybridization of human metaphase spreads with a rat cDNA probe specific for this enzyme and constructed from two clones previously characterized in our laboratory. The final construct had a 1.6-kb-long insert covering 92% of the coding sequence for GGT. The new insert was also freed of any GC tails introduced for the cDNA cloning, because we observed that these sequences were responsible for a high background. Using this probe for the analysis of 136 human metaphase spreads, we observed a strong specific signal on chromosome 22 at the interface of q111-112 and a minor peak in q131. Thus GGT might represent a new marker for the study of certain diseases which have chromosomal abnormalities at these loci.

gamma-Glutamyl transpeptidase (GGT) genomic sequences were isolated from rat and human libraries using a rat GGT cDNA as a cross-species hybridization probe. Characterization of the human GGT clones by restriction mapping clearly establishes that at least four different GGT genes or pseudogenes are present in the human genome. All the rat genomic clones cover a 12.5-kilobase sequence and exhibit a unique restriction pattern. A precise quantitation of the rat GGT gene copy number by Southern blot analysis demonstrates that this sequence is present as a single copy/rat haploid genome. Therefore, the GGT gene organization is different between rat and human species; this raises the possibility of different regulatory mechanisms in the two species.

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.

We localized gamma-glutamyltranspeptidase (GGT) in the rat pancreas by immunocytochemistry using the protein A-gold technique. The enzyme was found in the apical and zymogen granule (ZG) membranes of the pancreatic acinar cell. With ZG at the onset of exocytosis, labeling was seen over membrane, whereas content was unreactive. In the acinar lumen, the enzyme was generally associated with small vesicles previously described as "pancreasomes." This observation corroborates a recent proposal that a membrane-shedding process is associated with exocytosis in the exocrine pancreas. It also implies that some elements of the ZG membrane are not recycled after exocytosis. The cellular distribution of GGT was compared with GP2, another glycoprotein component of the ZG membrane, and differences in localization indicate different fates for these two proteins. Indeed, GP2 shows a strong signal with the basolateral membrane, whereas in the case of GGT the signal is barely detectable. The reverse situation is observed on the apical plasma membrane, GGT producing a much stronger signal than GP2. The failure to detect GGT in lysosomal structures, combined with the fact that some endocytic-like vacuoles in the vicinity of the apical plasma membrane give a positive reaction, supports the view that some GGT molecules are recycled in the ZG membrane after exocytosis. Our observations clearly demonstrate that a fraction of the protein components of the ZG membrane are not recycled after exocytosis, raising new questions regarding the concept of membrane recycling associated with regulated secretion.

The mechanism of rifampicin uptake and its relation with that of BSP were studied in isolated rat liver cells. The cells take up rifampicin by a saturable process independent of metabolic energy since antimycin A and potassium cyanide were without effect. The analysis of the uptake revealed two systems which both follow Michaelis-Menten kinetics: the first, with high affinity, displayed an apparent Kmof 0.13 ± 0.08 mM and an apparent Vmaxof 2.3 ± 1.2 nmoles/min/mg protein; the second, with much lower affinity, had an apparent Kmof 1.02 ± 0.46 mM and a Vmaxof 16.0 ± 5.9 nmoles/min/mg protein. Whereas rifampicin exhibited an apparently competitive inhibition on BSP uptake, the uptake of rifampicin was not inhibited by BSP. Moreover, no reciprocal counterflow was observed between these two drugs. These results suggest that rifampicin enters the liver cells by a carrier-mediated process independent of that previously reported for BSP uptake. It is concluded that several carriers are involved in the uptake of anionic drugs by rat liver cells.

Unconjugated sulfobromophthalein (BSP) inhibits state III respiration of rat liver mitochondria. It competitively inhibits the translocation into mitochondria of citrate, malate, phosphate and adenosine diphosphate, as studied by the inhibitor stop method. A double-beam spectrophotometric study strongly suggests that glutamate translocation is similarly inhibited. After perfusion of 65 mumol/hr/100 g for 90 minutes, bile flow is inhibited by 82% and liver adenosine triphosphate (ATP) falls by 60%. The amount of mitochondrial BSP can be computed form the amount of [35S] BSP still bound to mitochondria that are prepared at the end of such experiments; the amount of BSP lost during the isolation procedure is estimated from parallel experiments following binding of BSP in vitro. Comparison of the kinetic constants of mitochondrial transport and of their inhibition by BSP on the one hand and of liver concentration of substrates and BSP on the other gives rise to the conclusion that a strong inhibition of transports, mainly of phosphate, occurs in vivo and is responsible for the concomitant decrease in bile flow.

A comparative study of three commercially available blood filters for use in extracorporeal circuits was made in dogs. All filters were efficient in removing infused microclots from the circulation, but all caused mild thrombocytopenia and two produced minimal hemolysis. In dogs infused with microclots, only those animals without blood filters in the infusing circuit showed evidence of pulmonary microembolism at autopsy. It was concluded that while the filters have minimal disadvantages, their potential in reducing microembolism in extracorporeal circuits far outweighs these disadvantages.

The biosynthesis of gamma-glutamyl transpeptidase was investigated in hepatoma tissue culture cells. Pulse-chase experiments using [35S]methionine labeling have shown that the two glycosylated subunits of the enzyme (Mr = 58,000 and 29,000) derive from a single glycosylated precursor (Mr = 79,000 at early times). Only one polypeptide chain was immunoprecipitated from cell-free translation products and was shown to correspond to the nonglycosylated precursor (Mr = 64,000). Treatment with endoglycosidase H was used to probe for the transfer of the proteins from the endoplasmic reticulum to the Golgi and demonstrated: (i) that the precursor is at least partially cleaved in the endoplasmic reticulum; (ii) that part of the precursor is transferred to the Golgi where the processing of the oligosaccharide chains takes place. None of the precursor forms were detected at the surface of the cell where the mature enzyme was found. Tunicamycin, an inhibitor of protein glycosylation, did not prevent the proteolytic processing of the enzyme, but delayed the appearance of the mature enzyme at the cell surface. Monensin, which is known to alter Golgi functions, significantly delayed the acquisition of complex type oligosaccharides and the appearance of the enzyme at the cell surface. It did not, however, alter the proteolytic processing of the precursor of gamma-glutamyl transpeptidase. Taken together, these results show that gamma-glutamyl transpeptidase is synthetized as a single precursor which is at least partially cleaved in the endoplasmic reticulum. Part of the precursor is transferred to the Golgi where its oligosaccharide chains are processed.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.

Glucocorticoids are known to promote the formation of zymogen granules in acinar cells of the exocrine pancreas in vivo as well as in vitro. To gain insight into the mechanism of this regulation, we studied the effects of glucocorticoids on the synthesis of two components of the secretory granule membrane, the glycoprotein 2 (GP-2) and the gamma-glutamyl transpeptidase (GGT). It was demonstrated that following adrenalectomy, degranulation of pancreatic acinar cells is accompanied by a sharp decrease in GGT and GP-2 synthesis as measured by mRNA and protein accumulation. The decline of GGT synthesis was prevented by glucocorticoid replacement therapy, whereas GP-2 synthesis could be maintained with either glucocorticoid or estradiol treatment. These in vivo observations were corroborated and extended in an in vitro study using AR42J pancreatic cells. With this cell line, it was demonstrated that dexamethasone induces the formation of zymogen granules and the accumulation of a specific GGT transcript (mRNA III) by decreasing its degradation rate. At the same time, the GP-2 mRNA level was not modified by the hormonal treatment. These data demonstrate that glucocorticoids exert a positive control on the GGT expression in pancreatic cells at a post-transcriptional level. GGT, an enzyme of the glutathione metabolism, could play a significant role in protein packaging in secretory cells.

We have screened a cDNA library (20,000 clones) made from rat kidney poly(A)+ RNA, using an oligonucleotide probe that was a mixture of 14-base DNA oligomers containing all 32 possible sequences coding for residues 32-36 of the gamma-glutamyl transpeptidase (EC 2.3.2.2.) heavy chain. We isolated and sequenced two cDNAs corresponding to the mRNA coding for the entire length of the enzyme precursor. The nucleotide sequence that we obtained (2072 bases) reveals an open reading frame of 1707 nucleotides coding for the common precursor of both enzyme subunits. The amino acid sequence begins with the 21 residues located at the NH2-terminal hydrophobic region of the heavy subunit. We show that this sequence, which is not processed, is the only possible signal peptide in the sequence. Five potential N-glycosylation sites are present in the gamma-glutamyl transpeptidase sequence. Using one of the two cDNA clones as probe, a 2.2-kilobase sequence was detected by blot analysis in rat kidney and human fetal liver RNA.

gamma-Glutamyl transpeptidase (GGT) is an enzyme that plays a key role in interorgan glutathione transport. Three mRNAs (mRNAI, mRNAII, and mRNAIII) are known to encode the GGT precursor; they are initiated on three separate promoters on the single GGT gene. In this work, we identified by Northern blot and RNase H analysis a new GGT mRNA (mRNAIV). This mRNA differs from the others in its 5'-noncoding sequence. This mRNA species is the predominant GGT mRNA expressed in HTC hepatoma cells and in the small intestine in which its level increases from the base to the apex of the microvillus. The analysis of the GGT gene expression pattern in kidney, mammary gland, small intestine, liver, preneoplastic liver, and HTC hepatoma cells reveals a strong tissue or cell specificity. The mRNAIII was found in all the tissues and cells; in contrast, the expression of mRNAI, mRNAII, and mRNAIV is limited in normal tissues to the kidney and to the small intestine, the two tissues that display the highest enzyme activity. The synthesis of these three mRNAs is linked to the development of the kidney proximal tubule and to the differentiation of the enterocyte. The tissue and cell specificity of the GGT gene expression is based upon the use of multiple promoters that are controlled independently by specific cell factors.

gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit. The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments. We have investigated the first steps of GGT biosynthesis and processing in a cell-free system. mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis. In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme. Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000). This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H. Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain. This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.