52K Protein Research Articles

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.

Regional Distribution of 1 β-Hydroxysteroid Dehydrogenase in Rat Brain*

The activity and distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain is described. Oxidation of corticosterone to 11-dehydrocorticosterone was significantly increased by NADP; the reverse reaction was increased by NADPH. Cortisol was a poor substrate. Both 11-dehydrogenase (11-DH) and 11-oxoreductase (11-OR) activities were found in brains of rats from 6 days to adult, and 11-DH and 11-OR activities were positively correlated with each other. Highest enzyme activities were found in pituitary, cerebellum, hippocampus, and cortex. Lower levels were found in the olfactory region, hypothalamus, brain stem, preoptic nucleus, and amygdala. Two antisera to 11-DH, designated 56-125 and 56-126, reacted with a 34K component corresponding in mass to rat liver 11-DH on Western blots. The dominant species of protein in all brain regions reacting with rat liver 11-DH antibody 56-125, was at 26K mol wt. Antiserum 56-126 did not cross-react with the 26K protein. The 26K component was not a 34K degradation product. In each region of the brain, Western blot analysis showed that the 26K band intensity was directly proportional to enzyme activity. However, the 26K protein was devoid of 11-DH activity. All 11-DH and 11-OR activities were associated with the 34K antigen. The data demonstrate the nonuniform distribution of 11-DH in brain tissue. They are consistent with the notion that 11-DH may confer upon brain the ability to control intracellular levels of active glucocorticoids and in this way mediate steroid function within the cell.

The nucleotide sequence of RNA 2 of barley yellow mosaic virus

The complete nucleotide sequence of RNA 2 of a German isolate of barley yellow mosaic virus (BaYMV) has been determined. The RNA is 3585 nucleotides in length excluding a 3'-terminal poly(A) tail. The viral plus and minus strands in all three reading frames contained only one large open reading frame which started at positions 156 to 158 and terminated with a UAG codon at positions 2828 to 2830, thus encoding an Mr 98,000 polypeptide. Comparisons with sequences of other viruses revealed that the amino terminus of the polypeptide has homology with the proteolytic domain of the helper component proteinase of several potyviruses. It was determined that the 98K protein is a polyprotein composed of an amino-terminal 28K protein and a 70K protein.

Enteric adenovirus type 40: E1B transcription map and identification of novel E1A–E1B cotranscripts in lytically infected cells

Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Adl2 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A–E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4–5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A–E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A–E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.

Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato spotted wilt virus

Libraries of cloned cDNA were prepared from complete genomic RNA and isolated S RNA of the Bulgarian L3 isolate of tomato spotted wilt virus (TSWV-L3). Northern blotting of TSWV genomic RNA detected clones specific for the L, M and S RNAs in the library from complete RNA. S RNA-specific clones selected from both libraries covered approximately 2.8 kb (about 95%) of the S RNA. Sequencing of these clones showed TSWV-L3 S RNA to be ambisense. It contains two open reading frames (ORFs); one of 1401 nucleotides located on the viral RNA encodes an Mr 52,400 (52K) protein, and the other of 774 nucleotides on the complementary strand encodes an Mr 28,900 (29K) protein. Expression of the 29K ORF in bacteria and immunological analysis of the fusion protein synthesized confirmed that the 29K protein is the N protein of TSWV-L3. Comparison with the published sequence for the S RNA of a Brazilian TSWV isolate, CNPH1, revealed almost complete identity in the amino acid sequences for the 29K protein, but several clustered amino acid exchanges in the putative 52K protein. In addition, the separating non-translated intergenic region of the S RNA of the Bulgarian isolate is 81 nucleotides longer than that of CNPH1.

Differential Expression of Two Immediate Early Genes of Herpesvirus Saimiri as Detected by in Situ Hybridization

Transcripts from two immediate early (IE) genes have been identified in cells infected with the gamma herpesvirus, herpesvirus saimiri. One is a 1.3 kb RNA transcribed from the HindIII-G fragment of virus DNA (IE-G), the other is a 1.6 kb RNA from the gene for the IE 52K phosphoprotein. Labelled oligonucleotide probes specific for each of these RNAs have been used in in situ hybridization experiments to compare their expression in individual cells in infected populations. In the presence of cycloheximide, the IE-G RNA accumulates synchronously throughout the population of infected cells and prior to the asynchronous accumulation of RNA from the gene for the IE 52K protein in the same population of cells. This heterogeneity in the timing of expression of RNA from the IE 52K gene is paralleled by the asynchronous accumulation of the protein product. We conclude that transcription of the IE-G RNA is independent of expression of the IE 52K gene and that expression of the 52K gene requires (or is prevented by) factors which do not affect accumulation of the RNA from the IE-G gene.

Cathepsin D: A protease involved in breast cancer metastasis

Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Ad12 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.

Translation of cucumber necrosis virus RNA in vitro

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analysed in both rabbit reticulocyte lysate and wheatgerm extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of approximate Mr 34.6K was produced. In wheatgerm extracts, four proteins of approximate Mr values 41.6K, 34.6K, 24K and 20K were produced. The genomic locations of the CNV in vitro translation products were determined using several experimental approaches including, first, hybrid-arrested translation using negative-sense RNA corresponding to selected regions of the CNV genome, second, in vitro translation of synthetic positive-sense CNV transcripts and third, in vitro translation of CNV virion RNA fractionated according to size. Together these experiments demonstrated that the protein of Mr 34.6K is derived from the 5'-proximal coding region, the 41.6K protein is derived from an internal coding region, and that at least one but probably both the 24K and 20K proteins are derived from the 3'-terminal coding region. In addition, immunoprecipitation of in vitro translation products using anti-CNV polyclonal serum demonstrated that the 41.6K protein is the coat protein. The templates for the expression of CNV cistrons were investigated by in vitro translation of sucrose gradient-fractionated CNV virion RNA as well as in vitro translation of positive-sense synthetic transcripts.

Calcium binding protein (calbindin-D 28k) gene expression in the developing and aging mouse cerebellum

Calbindin-D 28k (CaBP 28k) protein and gene expression were examined in the mouse cerebellum during development and aging utilizing slot and Northern blot hybridization analyses for mRNA levels, Western blot analysis and radioimmunoassay (RIA) for protein levels, and by in situ studies using immunocytochemistry and hybridization cytochemistry on prepared tissue sections. Samples were obtained and analyzed from C57BL/6J mice aged day of birth and postnatal weeks 1, 2, 4, 8, and 120. A specific cDNA and antibody for CaBP 28k were utilized in these studies. Analysis of mRNA levels showed a steady rise in CaBP 28k mRNA from birth to a peak at postnatal week (3.4-fold increase) and then a decline to steady-state levels at postnatal weeks 4 and 8 (47% reduction of peak level) followed by a reduction of CaBP 28k mRNA to birth levels at postnatal week 120. The specificity of the changes observed was tested by reprobing blots with β-actin cDNA. Analysis of CaBP 28k protein levels by both Western blot and RIA showed a similar pattern. In situ analysis of CaBP 28k mRNA levels, based on hybridization signal (silver grains per cell), demonstrated a rise in cellular CaBP 28K mRNA levels which peaked at postnatal week 2 (416.9 ± 52.1) and then declined to steady-state levels by postnatal weeks 4 and 8 (267.4 ± 35.8). Cellular CaBP 28k mRNA levels exhibited a dramatic reduction in the aged cerebellum (postnatal week 120; 78.3 ± 16.0). The levels of cellular CaBP 28k mRNA corresponded to the intensity of immunoreactive CaBP 28k localized by immunocytochemistry. The results are consistent with the hypothesis that CaBP 28k may play a critical role in Purkinje cell maturation and maintenance. Decreased CaBP 28k gene expression in the aged cerebellum is not simply a consequence of cell loss (remaining viable cells exhibit reduced CaBP 28k gene expression). Thus, CaBP 28k may have a neuroprotective and maintaining the concentration of CaBP 28k in Purkinje cells may be important for their survival.

Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C

A Mr-80,000 acidic phosphoprotein ('80K protein') is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.

Immunodetectable Cytochromes P450 I, II, and III in Guinea Pig Adrenal—Hormone Responsiveness and Relationship to Capacity for Xenobiotic Metabolism*

Cytochrome P450s, proteins involved in metabolism of sterols, steroids, and a variety of foreign compounds, have been grouped into families based on amino acid sequence. We have identified a microsomal cytochrome P450 in guinea pig adrenal immunochemically related to P450c,d (P450I), induced in rat liver by methylcholanthrene. The inner zone localization, male predominance, and suppression by ACTH of this protein correspond to the ability of the adrenal microsomes to metabolize ethylmorphine. Its immunoreactivity, localization, and regulation distinguish it from P450(17) alpha (P450XVII) and P450(21) (P450XXI), known microsomal steroid hydroxylases. To examine whether other cytochrome P450s homologous to those in liver might be present in the guinea pig adrenal, microsomes were reacted with antibodies to hepatic P450s from families II and III. Each probe detected proteins in microsomes, but not in mitochondria, which were in the lower mol wt range of cytochrome P450s (47-50K). The immunoreactivity of all was diminished in animals treated with spironolactone, a compound which destroys cytochrome P450s in the adrenal, but not in liver. All were present in both outer and inner zones and in both males and females. None was suppressed by ACTH in the inner zone. Thus, only the previously described 52K protein reactive with anti-P450I corresponds in both distribution and ACTH response to the capacity for xenobiotic metabolism in guinea pig adrenal microsomes. On the other hand, unlike the 52K protein, the newly discovered proteins related to P450s II and III were all suppressed in the outer zone following dexamethasone treatment, suggesting that they might be related to dexamethasone-suppressible functions, such as metabolism of sterols and steroids.

Complete nucleotide sequence of clover yellow mosaic virus RNA

The entire genomic RNA of clover yellow mosaic virus was sequenced from cDNA clones and run-off cDNA transcripts. The genomic RNA is 7015 nucleotides in length [excluding a 3' poly(A) tail], with six open reading frames (ORFs) greater than 150 nucleotides in length. The first five ORFs encode proteins of Mr 191K, 26K, 12K, 6.5K and 28K, respectively. The sixth ORF lies completely within ORF1 and codes for a protein of Mr 14K. The capsid protein coding region (Mr 23K) is found within ORF5 which encodes the Mr 28K protein. Proteins encoded by ORFs 1 to 3 and ORF5 show strong homology with proteins of other potexviruses, especially papaya mosaic virus.

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein

We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: Staurosporine and K-252a

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8 R ∗, 9 S ∗, 11 S ∗)-(−)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2, 3, 9, 10-tetrahydro-8, 11-epoxy-1 H,8 H,11 H-2, 7b, 11-atriazadibenzo-[a, g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE ( ic 50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1–1000 nM) partially (<15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 μg/mL). Furthermore, prostaglandin E 2 (PGE 2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a ( ic 50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 ± 12.6 ng/10 6 cells) and immunoreactive leukotriene C 4 (LTC 4) (27.0 ± 2.6 ng/10 6 cells). Both histamine and LTC 4 release were inhibited by staurosporine and K-252a with an ic 50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca 2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.

Tumour Necrosis Factor Enhances induction by beta-Interferon of a Ubiquitin Cross-reactive Protein

Tumour necrosis factor alpha (TNF-alpha) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-alpha also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-alpha induction of UCRP occurred at the level of transcription. TNF-alpha induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-beta-interferon (IFN-beta) antibody. However no measurable increase in the mRNA specific for IFN-beta was detected after TNF-alpha treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-beta antibody indicates that these cells are making IFN-beta already. We conclude that the TNF-alpha induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-beta in the responding cells. Furthermore TNF functions to enhance the existing IFN-beta activity.

Identification of Two Genes in the Unique Short Region of Pseudorabies Virus; Comparison with Herpes Simplex Virus and Varicella-zoster Virus

We have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2.7 kb mRNA encoding the putative protein kinase and a 1.2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5' ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (greater than 95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mrs of 53K and 41K, and the 28K gene into a single protein with an Mr of 28K.

Transformation‐specific Decrease of Phosphorylation of 80K Protein, a Substrate of Protein Kinase C, in NIH3T3 Cells

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two‐dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c‐raf or H‐ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)‐stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N‐ras, K‐ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone‐inducible H‐ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation.

Simultaneous Bindings of ATPase Inhibitor and 9K Protein to F1F0-ATPase in the Presence of 15K Protein in Yeast Mitochondria1

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.

Cathepsin D in breast cancer

Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In vitro, pro-cathepsin D is an autocrine mitogen on breast cancer cells and can be auto-activated to degrade extracellular matrix and proteoglycans and to activate other proteinases in acidic microenvironments. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in lymph node-negative tumors. These results suggest that overexpression and possible derouting of cathepsin D plays an important role in invasion and metastasis of breast cancer.