Acrosin

The aim of our work is to evaluate the inhibition effect of some Allium extracts on acrosin and trypsin, comparatively with antioxidant effect and cytotoxicity of extracts. 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

The experiment with 0.5 g/mL of erode extracts of different parts of Allium plants (bulb, green leaf, white stalk) showed the inhibition of acrosin and trypsin activities, mostiy the red onion and garlic extracts. These results demonstrated that spermicidal effect of Allium extracts is determined also by their capacity of protease inhibition. The red onion extract shows the higher antioxidant capacity and a very low cytotoxicity. 

The aim of this work was to evaluate the inhibition effect of ome Allium aqueous extracts on acrosin and trypsin activities, a new aspect in the wide spectrum of Allium compound activities. The antioxidant capacity, probably responsible for antithrombosis and antitumoral effects, was determined for different parts of Allium plants (bulb, green leaf, white stalk). The toxicity of crude extracts was also determined. 

The values of acrosin activity obtained by the method based on the Kennedy et al. assay are summarized in Table 41.1. The boar semen ( 6.5 x 10 spermatozoa/mL) was treated, or not, with plant extract (0.5 g/mL) and the inhibition of acrosin activity indicates the spermicidal effect of plant extracts, about 50% for red onion extract and 25% for garlic extract, for instance. 

The micrographs obtained yiitkAPA assay show that treated spermatozoa with Allium extracts had low acrosin activity, especially the slides treated with garhc (Fig. 41.1a) and red onion extracts (data not shown). The untreated spermatozoa had protein-digested halos generated by acrosin activity on the gelatin substrate films (Fig. 41.1b). 

Table 41.1 Values of acrosin activity for untreated semen and Allium extracts treated semen...

Acrosin Sperm/PBS (1 0.1) Sperm/red onion Sperm/onion Sperm/garlic... 

Fig. 41.1 Micrographs of boar spermatozoa treated with Allium extraets (0.5 g/mL) (a) garlic bulb extract, (b) untreated spermatozoa. Formation of a clear halo around the sperm head indieated acrosin proteolytie activity...

ACONITATE DECARBOXYASE ACONITATE A-ISOM ERASE Acridine dye intercalation in DNA, SCATCHARD PLOT ACROSIN AcA,... 

The acrosome reaction is the loss of the acrosomal and plasma membranes in the acrosome region and the release of acrosin, hyaluronidase and other enzymes that disperse the cumulus complex and allow the sperm to penetrate the zona pellucida. After capacitation and the acrosome reaction, sperm penetrate the extracellular cumulus matrix and bind with zona protein 3, a heavily glycosylated protein of the zona pellucida. The first segment of the sperm to make contact with the oolemma is usually the inner acrosomal membrane, followed by the postacrosomal region. The plasma membrane of the sperm attaches to microvilli on the oolemma. Sperm-egg fusion is apparent from reduced movement of the sperm tail (Yanagimachi, 1970,1988 Takano et al.,... 

Takano H, Yanagimachi R, Urch UA (1993) Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with the oolemma Inhibitor studies using the golden hamster. Zygote, 1 79. 

Gossypol (dimeric sesquiterpene) Gossypium hirsutum, Montezuma speciosissima (Malvaceae) Acrosin, Azocoll proteinase [110, 111]... 

Structure—Function Properties of the Sperm Enzyme Acrosin... 

Contemporary studies on the ZP lysin acrosin were perhaps initiated with the observation that synthetic protease substrates were hydrolyzed by sperm extracts (4) and that the enzyme could be effectively extracted from sperm at pH 3 (5). The enzyme has now been purified from several species, shown to be specific for Arg/Lys peptide bonds, and to be a serine active site endoprotease. In 1972, Meizel provided evidence that a zymogen form of acrosin existed, termed proacrosin (6.). 

No sperm enzymes have yet been described which meet all of these criteria. However, acrosin satisfactorily meets the first three criteria and, with the identification of the zona pellucida glycoprotein which is a substrate for acrosin, it is now possible to approach experimentally the fourth criterion (7). 

HEDRICK ETAL. Structure—Function Properties of Acrosin... 

Proacrosin and acrosin are glycosylated. Rabbit proacrosin and porcine acrosin are 8% carbohydrate by weight (8,16.). Porcine acrosin has an N-linked oligosaccharide at Asn3 (17). Data for... 

Mr fragment (light chain) is generated which remains bound to the remainder of the acrosin molecule (heavy chain) by disulfide bonds, regardless of whether the proacrosin is the 62K Mr size variant isolated from testis or the 55K Mr or 43K Mr size variants isolated from spermatozoa (15). These latter results are presently the only direct evidence for a proacrosin activation mechanism involving limited proteolysis of a single polypeptide chain to produce a two-chain acrosin. 

The sequences of porcine and caprine acrosin light chains and the N-terminal sequences of the acrosin heavy chains are shown aligned with the sequences of several serine proteases in Figure 1. Sequence similarity between porcine and caprine acrosins is readily apparent, as is the similarity of the acrosin sequences (range of... 

Acrosin heavy chain (po) Acrosin heavy chain (ca) Plasmin (hu)... 

Figure 1. Sequences of acrosin light chains and N-terminal sequences of acrosin heavy chains shown aligned with the sequences of corresponding portions of other serine proteases. Positions identical with the porcine acrosin sequence are underlined. Alignments for plasmin, prothrombin, Clr b-chain, cathepsin G, and plasminogen activator are from (24), except minor adjustments were made to maximize similarity with acrosin. Sequence similarities expressed as percent positional identity to the porcine sequences are indicated to the right of each sequence. Abbreviations po - porcine, ca - caprine, bo - bovine, hu - human.

PROTEOLYTIC ACTIVITY. In comparison with pancreatic trypsin, acrosin is 50% larger, contains carbohydrate, and is more hydrophobic (11). Although acrosin has often been referred to as trypsin-like, the above structural differences are reflected in kinetic and hydrolytic specificity differences, in different interactions with membranes, and in the newly described lectin function of acrosin. 

Substrate specificity differences between boar acrosin and trypsin are not particularly manifest when using small substrates, but these enzymes show distinctly different kinetics of porcine ZP hydrolysis (34). The loss of 30% mass in the conversion from m - to m -acrosin has little effect on the kinetic analyses of inhibition and substrate preference with artificial substrates and small trypsin inhibitors, indicating that this excised portion of the enzyme contributes little to the topography of the active site (35). From Km analyses with amide and ester substrates of Arg and Lys, acrBSin prefers the Arg substrates over Lys, and Km differences between amide and ester substrates indicates that ac Ssin proceeds kinetically through a classical double displacement mechanism as does trypsin (36). 

Acrosin causes the limited and specific cleavage of only certain portions of the ZP (7,32,38) The ZP is conq>osed of three families of glycoproteins, denoted 90K, 55K and 55K, and two derived components, 65K and 25K (39). The f>5K and 25K components are produced from the 90K family by a proteolytic event in the follicle. The 90K family, and the 90K and 65K components, were specifically... 

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