Identification and Activation of Profollipsin, a Latent Precursor form of Porcine Follipsin

Abstract

A latent protease has been identified in column fractions obtained during the purification of the porcine ovarian serine protease follipsin. The latent enzyme was readily activated by trypsin treatment. The trypsin-activated enzyme was purified using a benzamidine-Sepharose 6B column and was shown to be composed of two distinct, covalently associated polypeptides with M , of 45000 and 32000. This polypeptide chain composition, together with its substrate specificity, inhibition profile using various protease inhibitors, cross-reactivity with anti-follipsin antibody, and ability to activate single-chain precursor tissue plasminogen activator, indicated its identity as porcine follipsin. The activation of the enzyme with trypsin was found to occur by the hydrolysis of an internal peptide bond resulting in a two-chain structure. Thus, we conclude that the latent enzyme is the inactive precursor form (profollipsin) of follipsin. The present study also shows that the follicular fluid of porcine ovary contains a profollipsin-activating enzyme activity. We have previously identified an additional enzyme activity in the follicular fluid of porcine ovaries that is capable of hydrolyzing synthetic, arginine-containing peptide 4-methylcoumaryl-7-amide (-NH-Mec) substrates. Purification and characterization studies revealed that the enzyme follipsin is a serine protease structurally similar to human plasma kallikrein [ 14, 151. Subsequently, the enzyme has been shown to efficiently convert human single-chain precursor tissue-type plasininogen activator (sctPA) to its mature form (tPA) in vitro [16].Follipsin consists of two distinct polypeptide chains with M , of 45 000 and 32 000, associated covalently, and is thought to be synthesized as a single polypeptide chain precursor protein [IS]. The question is thus raised as to where the precursor enzyme undergoes conversion to active two-chain follipsin. In an attempt to approach this problem, we noted that trypsin treatment of certain fractions, which were obtained in the column chromatoCorve,~poi?tl~,rzae fo T. Takahashi,

Identification of Components of the Intrafollicular Bradykinin-Producing System in the Porcine Ovary1

“…We confirmed our previous observation [10,21] that the ovarian follicular fluid contains an enzyme capable of activating prePK. In order to determine its chemical makeup, we purified the enzyme by a series of column chromatographic steps on CM-52, DE-52, and benzamidine-Sepharose 6B columns (Fig.…”

“…Enzyme activity of ovarian follicular fluid prePK-activating enzyme was assayed as previously described [10]. Briefly, aliquots of the fractions were incubated at 37ЊC in a volume of 32 l in 0.1 M Tris-HCl (pH 8.0) with 5.6 pmol prePK for 5 min.…”

“…Our subsequent studies revealed that plasma kallikrein (PK) is structurally and functionally homologous to porcine follipsin [9,10]. These findings prompted us to further investigate the biological significance of the presence of intrafollicular PK using porcine ovaries.…”

Topically applied fatty acids are elongated before incorporation in the stratum corneum lipid matrix in compromised skin

“…Subsequently, FA 18 is further elongated by ELOVL1 and ELOVL4, to chain lengths up to C28:0, and >C30:0, respectively. 35,36 Our results suggest that ELOVL1, ELOVL6, and possibly ELOVL4 are able to elongate not only endogenous protiated FAs but also topically applied (partially) deuterated exogenous FAs. Furthermore, the results suggest that the elongation of FA 16 by ELOVL6 is the rate limiting step, because FA 16 is present but the elongation products with 18 and 20 are not detected.…”

Symmetry Information Inherent in Powder Diffraction Data: α- and β-Quartz as Examples

“…The positions of the peaks at (0.25,0,0.17) in both P(uOw)'s are also slightly different because of the displacement of the atoms. Since only small ripples except the high peak The sections P(uOw), P(uvO) and P(uv~) of the composite Patterson function were evaluated using the intensity data of a single crystal measured by Kihara (1990) (Fig. 4).…”