Kidney Tumor Cells Research Articles

Cloning and sequence analysis of the cDNA encoding P-450 aromatase (P450arom) from a rainbow trout (Oncorhynchus mykiss) ovary; relationship between the amount of P450arom mRNA and the production of oestradiol-17 beta in the ovary.

The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17 beta by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic. Northern blot analysis revealed 2.6 kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-beta production by the ovarian follicles, oestradiol-17 beta production may be regulated, in part, by the amount of P450arom mRNA present.

Tissue-specific expression of Na,K-ATPase β-subunit Does β2 expression correlate with tumorigenesis?

We have found a substantial decrease in the level of Na,K-ATPase β2-subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of β1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of β1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of α1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of β2-subunit in the process of tumorigenesis as was shown for some other adhesion molecules.

3β-Hydroxysteroid dehydrogenase/Δ 5→4-isomerase expression in rat and characterization of the testis isoform

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3β-hydroxysteroid dehydrogenase/Δ 5→4-isomerase (3β-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3β-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3β-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3β-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD +, but not NADP +, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent K m values of 0.13 and 0.09 μM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3β-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3β-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3β-HSD content, a 3β-HSD is also expressed in liver in a sex-specific manner.

Urinary protein C inhibitor. Glycosaminoclycans synthesized by the epithelial kidney cell line TCL-598 enhance its interaction with urokinase

Protein C inhibitor (PCI), also known as plasminogen activator inhibitor 3, inhibits a variety of serine proteases by forming sodium dodecyl sulfate-stable 1:1 complexes. In purified systems PCI is only a weak inhibitor of urokinase. Nevertheless, complexes between PCI and urokinase are found in appreciable amounts in native human urine. Since PCI activity is stimulated by heparin and other glycosaminoglycans, we investigated the presence of stimulating glycosaminoglycans on cells lining the urinary tract. We chose the epithelial kidney tumor cell line TCL-598 as a model and isolated metabolically labeled glycosaminoglycans. TCL-598 incorporated [35S] sulfate into high Mr components (Mr greater than 200,000 and approximately 75,000) as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of cell extracts; the Mr greater than 200,000 component bound specifically to PCI-Sepharose 4B and was eluted either with heparin (5 mg/ml) or with NaCl (2.0 M). Treatment of this PCI-binding material with chondroitinase ABC, but not with chondritinase AC or heparitinase, abolished binding to PCI-Sepharose, confirming the glycosaminoglycan nature of this material and suggesting the involvement of dermatan sulfate in binding. These glycosaminoglycans eluted from PCI-Sepharose stimulated urokinase inhibition by PCI in a dose-dependent way and enhanced complex formation of 125I-urokinase and PCI as did in control experiments dermatan sulfate from porcine skin and from bovine mucosa. Our results suggest that PCI activity might be regulated also in vivo by the presence or absence of stimulating glycosaminoglycans; dermatan sulfate-containing glycosaminoglycans associated with kidney cells might be responsible for stimulation of the urokinase inhibitory activity of PCI in the urinary tract; the type of glucosaminoclycans might furthermore regulate enzyme specificity of PCI.

Correlations between natural resistance to doxorubicin, proliferative activity, and expression of P-glycoprotein 170 in human kidney tumor cell lines

The natural resistance to doxorubicin of 15 human renal carcinoma cell lines was analyzed and compared to proliferative activity and expression of P-glycoprotein. We found a significant negative correlation between proliferative activity and natural resistance to doxorubicin, as well as between proliferative activity and the expression of P-glycoprotein. A positive correlation between resistance and expression of P-glycoprotein was found.

Continuous production of prourokinase in feed harvest and perfusion cultures

The production of prourokinase (PUK) by a human kidney tumor cell line is studied in long term cultures. Cells are grown on microcarriers which are retained inside the reactor by sedimentation or with a spin filter. Two modes of operation are compared: feed harvest at an average medium exchange rate of 0.3 d(-1) and continuous perfusion at a higher dilution rate of 1.5 d(-1). In the two systems a stable production of PUK has been maintained for more than 400 h. Kinetics of cellular growth, nutrient consumption, and metabolite and PUK excretion are similar. After an initial rapid growth period, one observes a 10-fold reduction in all the cellular metabolic activities during the stationary phase. Continuous perfusion yields a higher cell density (7 x 10(6) cells.mL(-1)) than feed harvest (3 x 10(6) cells.mL(-1)), which results in a twofold increase in the reactor productivity. But higher final enzyme activities, about 250 ru.mL(-1), are obtained in the feed harvest recovered medium than in the perfusion medium, 100-150 ru.mL(-1). The cumulative medium consumption per mass of product is the same in the repeated batch and in the continuous operation mode.

Modulation of vincristine sensitivity of human kidney tumor cells by pharmacological agents interfering with intracellular signals. No apparent relationship to changes in cytoplasmic Ca 2+ or pH

The effect of substances proposed to modulate intracellular signal systems on growth and sensitivity to vincristine in the human kidney tumor cell line ACHN was investigated and related to changes in cytoplasmic free Ca 2+ concentration ([Ca 2+] i) and cytoplasmic pH (pH i). Presence during culture of the protein kinase C (PKC) activator 12- O-tetradecanoyl phorbol 13-acetate (TPA) had no effect on cell growth but significantly increased the EC 50 concentration for vincristine inhibited cell growth. There was no indication for endogenous PKC activity being responsible for basal vincristine insensitivity since it was not affected by the PKC inhibitor H-7. The Ca 2+ ionophore ionomycin tended to increase cell growth and induced vincristine resistance, whereas the calmodulin inhibitor W-7 had opposite effects. Presence during culture of the adenylate cyclase activator forskolin did not affect basal cell growth but dose-dependently made the cells more sensitive to vincristine. The modulators of vincristine sensitivity had no immediate effect on pH i, whereas after 3 days of incubation ionomycin and forskolin tended to increase pH i. Ionomycin and forskolin induced an immediate increase in [Ca 2+] i which remained after 3 days only for ionomycin, whereas TPA decreased [Ca 2+] i, a change which tended to remain after 3 days of incubation. It is concluded that perturbation of the intracellular signal system may affect both cell growth and cytotoxic drug sensitivity. However, there is no apparent relationship between immediate or late changes in [Ca 2+] i and pH i and vincristine sensitivity.

Human 3β-Hydroxysteroid Dehydrogenase/ δ5→4Isomerase from Placenta: Expression in Nonsteroidogenic Cells of a Protein that Catalyzes the Dehydrogenation/Isomerization of C21 and C19 Steroids*

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

Verapamil and cyclosporin a sensitize human kidney tumor cells to vincristine in absence of membrane P-glycoprotein and without apparent changes in the cytoplasmic free Ca2+ concentration

Vincristine (Vcr) dose dependently inhibited growth of the kidney adenocarcinoma cell line ACHN during 4 days of culture. Verapamil (Ver) at 10 microM and cyclosporin A (CsA) at 1 microgram/ml had no effect on cell growth but significantly potentiated the action of Vcr, despite the absence of the multidrug resistance associated membrane P-glycoprotein (P-gp). Neither Ver nor CsA had any acute or long term effects on cytoplasmic free Ca2+ concentration (Ca2+i), except for a small Ver induced increase after 36 h of incubation. The results indicate that Ver and CsA may have a sensitizing effect on chemotherapeutic drug sensitivity also in absence of P-gp. However, these effects are probably not mediated by changes in Ca2+i.

Atrial natriuretic factor effects on cyclic nucleotides in a human renal cell line.

The natriuretic effects of atrial peptide hormones have been attributed, at least in part, to their stimulation of guanylate cyclase activity in renal cell membranes. The effects of atrial natriuretic factor (ANF) on stimulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) accumulation were investigated in cloned human kidney tumor (hKT) cells and parent cells from a human renal tumor epithelial cell line (SK-NEP-1). Human ANF-(99-126) (10(-6)M) stimulated (p less than 0.001) cellular cGMP accumulation in a dose-dependent manner from a basal level of 0.26 +/- 0.04 to 3.73 +/- 0.81 pmol/mg protein/5 mi (mean +/- SEM, n = 13). ANF stimulation of cGMP accumulation was specific, in that high concentrations (10(-6)M) of atriopeptin I [rat ANF-(103-123)], angiotensin II, arginine vasopressin, and amiloride (10(-4)M) did not increase basal cGMP. Amiloride (10(-4)M) enhanced (p less than 0.01, n = 6) the ANF stimulation of cGMP accumulation (1.24 +/- 0.39 pmol/mg protein/5 min), particularly at low doses of ANF (10(-10)M) where stimulation by ANF without amiloride (0.34 +/- 0.08 pmol/mg protein/5 min) was barely distinguishable from a basal level (0.19 +/- 0.02 pmol/mg protein/5 min) of cGMP accumulation. The stimulatory effect of ANF (1.59 +/- 0.07 pmol/mg protein/5 min) was attenuated (0.75 +/- 0.06 pmol/mg protein/5 min, p less than 0.01, n = 6) by preincubation of the cells with pertussis toxin but not by cholera toxin. ANF (4.56 +/- 0.93 pmol/mg protein/5 min, n = 8) did not affect cAMP accumulation (4.32 +/- 0.98 pmol/mg protein/5 min) in hKT cells. This is the first report of an ANF responsive human renal cell line, and its use should facilitate investigation of ANF-receptor interactions.

The regulation of oestrogen biosynthesis in human adipose tissue

SynopsisThe enzyme complex responsible for the conversion of androgens to oestrogens is called aromatase. This enzyme is expressed in a number of tissue sites, including granulosa cells, placenta, hypothalamus and adipose. Oestrogen formation in adipose tissue has been implicated in the aetiology of endometrial and breast cancer. Aromatase comprises two components: a member of the cytochrome P-450 gene family known as aromatase cytochrome P-450, and a flavoprotein, NADPH-cytochrome P-450 reductase. We have recently obtained a full-length cDNA insert encoding human aromatase cytochrome P-450 and have expressed it in COSI monkey kidney tumour cells. The resulting transcript encodes a protein which is capable of converting androstenedione, testosterone and 16α-hydroxyandrostenedione to the corresponding oestrogens. Furthermore, characterisation of the aromatase cytochrome P-450 gene indicates that in humans there is only one such gene, which spans about 30kb and has at least nine exons. From these results we conclude that there is only one aromatase enzyme in humans. Study of the expression of this enzyme in human adipose stromal cells indicates that it is subject to regulation by a number of factors including cAMP, phorbol esters, and the growth factors, EGF, TGF-α, TGF-β, TNF and 1Lβ. Since several of these growth factors are produced by breast cancer cells in response to oestrogens, the possibility exists for paracrine and autocrine feedback loops within the breast, in which the growth of a tumour is regulated by oestrogen produced locally in the adipose cells of the surrounding tissue, and this in turn is regulated by the growth factors produced by the tumour as a result of oestrogen action.

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.

The effect of 3-deazauridine and dipyridamole on uridine utilization by mice

The inhibition of uridine utilization by 3-deazauridine, an inhibitor of uridine kinase, and by dipyridamole, an inhibitor of the facilitated transport of nucleosides was examined. 3-Deazauridine (500 mg/kg) markedly inhibited (>70%) the formation of uracil nucleotides from uridine in liver, kidney, and L1210 tumor cells. The degree of inhibition is greatly reduced by 6 hr after administration of the drug. Dipyridamole (100 mg/kg) did not significantly reduce salvage of uridine by liver or kidney and produced only small, transient reductions in salvage by L1210 tumors. Dipyridamole pretreatment did not alter the rate of clearance of uridine from the plasma.

A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas.

The BALB/cf/Cd substrain of mice, developed by inbreeding and selection, shows a 70% incidence of spontaneous kidney adenocarcinomas. Initially foster-nursed on C3H mothers, these mice no longer produce mammary tumors, but there is evidence that mouse mammary tumor virus (MMTV) is involved in the formation of these renal carcinomas. We identified a chromosomal region called int-41, representing a locus interrupted by the integration of an exogenous MMTV provirus in a BALB/c mammary tumor. We found a DNA rearrangement and transcriptional activation in the domain specified by the int-41 probe in one primary kidney adenocarcinoma. A 5.2-kb int-41 specific mRNA was detected in the kidney tumor cell line established from a transplantable renal adenocarcinoma. This mRNA hybridized with MMTV and int-41 specific probes suggesting that it is a hybrid molecule composed of MMTV-LTR sequences covalently linked to host cell RNA. This mRNA was strongly stimulated by the presence of glucocorticoid hormone in the culture medium. Our data are compatible with the hypothesis that the int-41 chromosomal domain is involved in the neoplastic transformation of epithelial cells from different organs.

Carcinogenicity of catechol estrogens in Syrian hamsters

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters ( 4 5 and 4 5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol ( 0 5 ) or 2-methoxyestradiol ( 0 6 ) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol > 4-hydroxyestradiol > 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.

Correlation of 19F-NMR spectra of halothane in rat tumor and non-tumor tissues with membrane alterations

The membrane environments in normal and tumor rat tissue and the effect of hyperthermia thereon are studied with 19F-NMR spectroscopy of the general anesthetic halothane. Normal and tumor cell types are clearly differentiated by the halothane resonance. A hydrophobic environment prominent in tumor tissue is more sensitive to heat treatment than the corresponding environments of normal cells. Studies of extracted lipids suggest that this may be due in part to the considerable difference in lipid temperature response which exists between normal and kidney tumor cells.

Biosynthesis of the adenosine deaminase-binding protein in human fibroblasts and hepatoma cells.

The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.

Secreted glycoproteins of human kidney tumour cells contain sulphated complex-type oligosaccharides.

Human kidney tumour cells in culture incorporated [3H]glucosamine and 35SO4 into glycoprotein products, which were secreted into the culture medium. The effects of sodium butyrate, a known differentiation-inducing agent, on the production of these sulphated glycoproteins were studied. Cells were cultured in the absence or presence of butyrate (2 mM) in serum-containing medium, for various times, and the labelled glycoproteins were partially purified by DEAE-cellulose chromatography. Treatment of these cells with butyrate resulted in an increase in the synthesis of secreted [3H]glucosamine- and 35SO4-labelled glycoproteins over several days of culture. This same increase in levels of 35SO4 incorporation was not observed with B16 melanoma cells. Sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis revealed that five major glycoproteins labelled with [3H]glucosamine also were labelled with 35SO4. The major secreted glycoproteins from cells cultured in the absence or presence of butyrate over a 3-day period were similar by SDS/polyacrylamide gel electrophoresis. Analyses of Pronase-derived glycopeptides indicated that these secreted 3H/35S-labelled glycoproteins contained sulphated oligosaccharides with terminal sialic acid----Gal----GlcNAc residues similar to glycoproteins secreted by vascular endothelial cells.

Large scale isolation, purification, and partial characterization of the intermediate filament-specific, Ca 2+-activated proteinase from porcine kidney and Ehrlich ascites tumor cells: A comparative study

The Ca 2+-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH 4) 2SO 4 saturation, followed by hyroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca 2+-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca 2+-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W. J. Nelson and P. Traub, (1983) J. Biol. Chem. 257, 5544–5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined.

A Mitochondria1 Protease that Cleaves the Precursor of Ornithine Carbamoyltransferase

Ornithine carbamoyltransferase of rat liver mitochondrial matrix (subunit Mr = 36,000) is synthesized extra-mitochondrially as a larger precursor (subunit Mr = 39,400) which is transported into mitochondria, in association with post-translational proteolytic processing. Rat liver mitochondria convert the precursor to the mature enzyme as well as to a 37,000-Mr product, a possible intermediate of the processing [Mori, M., Miura, S., Tatibana, M., and Cohen, P.P. (1980) Proc. Natl Acad. Sci. USA, 77, 7044-7048]. A protease responsible for the conversion of the precursor to the 37,000-Mr product was purified 140-fold from the matrix fraction of rat liver mitochondria. The protease had an estimated Mr of 108,000 and an apparent pI of 5.5. Mature ornithine carbamoyltransferase (0.5 microgram) did not inhibit the cleavage of the precursor by the protease and presumably the latter cleaves a specific site on the extrapeptide of the carbamoyltransferase precursor. The protease was inhibited by metal-chelating reagents such as EDTA, o-phenanthroline and zincon and by a high concentration (1 mM) of leupeptin. It did not cleave several of the protein and peptide substrates tested including the precursor of mitochondrial carbamoyl-phosphate synthetase I. Apparently the same protease activity is widely distributed among mitochondria of rat kidney, spleen, heart and ascites tumor cells, all of which lack ornithine carbamoyltransferase. A possible physiological role of the protease in the processing of the mitochondrial protein precursors is discussed.