Prolyl 4‐hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4‐hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta‐subunit of prolyl 4‐hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non‐coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta‐subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4‐hydroxylase tetramer.

Immunization with a peptide fraction from ovarian cancer urine resulted in an antiserum that reacts with a 6,000 dalton (6K) peptide. In urine from some patients with advanced gynaecological cancer, the concentration of 6K peptide was high enough to be detected by immunodiffusion. By radioimmunoassay the levels in normal serum could be determined. The concentrations in serum was 5-20 microgram/l and that in urine 5-50 microgram/l. Elevated levels were observed in urine from 5 out of 8 patients with ovarian cancer, 10 out of 14 patients with cervical cancer and 9 out of 14 patients with endometrial cancer. The highest level observed was 12,000 microgram/l. High concentrations of the 6K peptide (100-300 microgram/l) were observed in amniotic fluid from 14-16 weeks of pregnancy. Some tumor extracts also had a high concentration of the 6K peptide. No immunological relationship was observed between the peptide and known tumor-associated antigens or serum proteins. These results suggest that the peptide is a new oncodevelopmental peptide which may have significance as a tumor marker.

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.

Placental protein 12 (PP12) was originally isolated from term human placenta and adjacent membranes. Recently we found that the site of PP12 synthesis is decidua but not placenta. In this work, the purity of PP12 was first tested by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis and by reverse phase HPLC, and the N-terminal amino acid sequence of 15 residues was determined by a liquid-phase sequencer. A single amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala-Asp-Glu-Leu-Ala-Leu was obtained showing identity to the known N-terminal amino acid sequence of somatomedin-binding protein from human amniotic fluid. Like the latter, PP12 bound somatomedin (insulin-like growth factor I) as demonstrated in gel chromatography by a shift in the elution pattern of [125I]iodo-insulin-like growth factor I after incubation with PP12. These data show that PP12 is a somatomedin-binding protein and extend through previous literature on PP12 the existing knowledge on the physiology and pathophysiology of somatomedin-binding protein(s) in human reproduction and cancer.

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