Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

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Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the A PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyruvate phosphate dikinase contains a pivotal histidyl residue which functions to mediate the transfer of phosphoryl moieties during the reaction catalyzed by the enzyme. The tryptic peptide which contains this essential histidyl residue has been isolated by a two-step procedure originally developed by Wang and co-workers [Wang, T., Jurasek, L., & Bridger, W. A. (1972) Biochemistry 11, 2067]. This peptide has been sequenced by the manual dansyl-Edman procedure and is shown to be NH2-Gly-Gly-Met-Thr-Ser-His-Ala-Ala-Val-Val-Ala-Arg-CO2H. There is no readily interpretable homology between this peptide and other phosphorylated histidyl peptides previously isolated from other enzymes. By use of Chou & Fasman [Chou, P. Y., & Fasman, G. D. (1974) Biochemistry 13, 222], it is predicted that the sequence contains an alpha helix from the methionine residue through to the carboxyl terminal arginine residue.

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Seven Merino-Border Leicester cross-bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the alpha subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P less than 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n = 5) or had been immunized with 300 micrograms KLH (n = 4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin-binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in-vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin alpha subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.

It is shown that polyphosphates are not generated in significant amounts in the phosphoglycerate kinase reaction; polyphosphate is more effective than ATP in the formation of glucose 6-P by glucokinase, but the rate with ATP may be adequate to meet the requirements of glucose metabolism; PP1 is far more effective than ATP as a phosphate donor in the formation of fructose 1,6-P2 by phosphofructokinase; PPj rather than ATP almost certainly is used in this reaction; and, aside from glucokinase and phosphofructokinase, the enzymes of phosphorylation are specific in their requirements of phosphate donors or acceptors and are present in adequate amounts to meet the requirements of glucose metabolism by the propionic acid bacteria.The propionic acid bacteria are unusual in that they utilize PP, in several phosphorylation reactions in place of ATP (1, 2). (5) have shown that they form poly(P) from ATP (Reaction 5).polyphosphate glucokinase glucose + poly(P)nglucose 6-P + poly(P),,1 [4] polyphosphate kinase ATP + poly(P),', ADP + poly(P),+1 [5] Of special interest is the report by Uryson and Kulaev (3) that Propionibacterium freudenreichii has poly(P) glucokinase activity but only a trace of ATP glucokinase activity.This finding raises the possibility that P. freudenreichii phosphorylates glucose almost exclusively with poly(P). In addition, Kulaev et al. (4) MATERIALS AND METHODSCultures and Conditions for Growth. Propionibacterium arabinosum, ATCC 4965; P. freudenreichii, ATCC 6207; and P. shermanii, 52W were investigated. The latter was originally isolated by Virtanen (8) and was later given the number 52W by C. H. Werkman (Iowa State University). The bacteria were grown at 30°C in 15 liters of medium containing glucose, 45 g; Na2CO2, 82.5 g; KH2PO4, 153 g; yeast extract, 76 g; Co(NO3)266H2O, 0.15 g; thiamin HCl, 15 mg; calcium pantothenate, 15 mg; biotin, 7.5 mg; and 20 ml of trace elements (boric acid, 75 mg; CuS04, 6 mg; Nal, 13.5 mg, FeSO4, 66.2 mg; ZnCl2, 28.4 mg; and SeO2, 16.6 mg per 100 ml). Glucose and Na2CO3 were each sterilized separately. Glycerol medium was identical except 450 g of glycerol was substituted for glucose. Lactate medium contained (per 15 liters) sodium lactate (USP 60% syrup, Pfansted Laboratories, Worthington, IL), 750 ml; yeast extract, 60 g; tryptone, 30 g; K2HPO4, 60 g; NaH2PO4, 30 g; FeSO4, 0.15 g; and CoNO3, 0.15 g; vitamins and trace elements were as given above for the glucose medium. Bacteria were harvested using a Sharples centrifuge, either after 4-6 days during the stationary phase of growth (Table 1) or after 2, 4, and 7 days of growth (Table 2). A portion of the 15 liters of culture was removed and the gas phase was restored with CO2 after each removal to maintain anaerobic conditions. In the experiments of Tables 1 and 2, the packed cells were held at -70°C until preparation of the crude extract.Crude Extracts and Fractionations. The cells were broken by passage through a French press or by grinding in an Ep- 312The publication costs of this article were defray...