Functional properties of caltrin proteins from seminal vesicle of the guinea pig.

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.     

Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig

Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.     

Purification and initial characterization of guinea pig testicular acrosin

Guinea pig (GP) acrosin was purified following acid extraction of testicular acetone powder, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion-exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose. Final purification was achieved by re-chromatography on Sephadex G-100. Enzymatic activity was detected by following the hydrolysis of N-benzyloxycarbonylarginyl amide of 7-amino-4-trifluoromethylcoumarin at 37 degrees C, pH 8.0, before and after activation. GP testicular acrosin exhibited a molecular weight of 48,000 by gel filtration and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE in gels containing 0.1% gelatin, protease activity was observed to comigrate with the major protein detected by silver staining. The purified GP acrosin showed cross-reactivity with a monospecific polyclonal rabbit antiserum directed against boar sperm acrosin and exhibited reversible pH-dependent activation. The physiochemical characteristics of the purified protein, including the amino acid composition, resemble those reported for acrosins from other species.     

高等哺乳动物LEM结构域蛋白家族的研究进展

在高等动物细胞开放式有丝分裂过程中,细胞核膜会发生高度有序的周期性去组装和装配的动态变化。近年的研究结果表明是LEM家族蛋白成员通过与BAF因子相互作用介导了内核膜、核纤层蛋白以及染色体之间的相互作用。LEM蛋白、核纤层蛋白以及BAF因子直接相互作用形成的三元复合体在结构与功能上是相互依赖的,在此结构与功能上组成的网络体系是形成细胞核的一些基本生物学过程的重要条件。该复合体在调控有丝分裂M期后期和末期染色体的正常分离、有丝分裂后核膜的重组装,细胞分裂间期细胞核及核膜形态维持,调控DNA复制和DNA损伤修复,调节基因表达和信号通路以及逆转录病毒感染等方面发挥着重要的生物学功能。并且LEM蛋白相关基因的异常对核纤层疾病和肿瘤的发生发展具有重要的影响。文章主要针对LEM蛋白家族成员的结构以及功能研究进展进行了详细的综述。     

Proacrosin activation and acrosin release during the guinea pig acrosome reaction

The kinetics of proacrosin activation and release from guinea pig spermatozoa during the nonsynchronous acrosome reaction were studied. Epididymal spermatozoa were incubated at 37 degrees C in a defined medium (pH 7.8) containing 1.7 mM Ca2+. After 195 min, 78% of the motile spermatozoa had undergone the acrosome reaction as determined by light microscopy. Acrosin and proacrosin levels in the spermatozoa and medium were measured at the beginning of the incubation period. Most of the total acrosin activity (78%) was associated with the spermatozoa, of which greater than 90% was in the form of proacrosin. Proacrosin represented a small, stable fraction (23%) of the total acrosin in the medium; it did not activate to acrosin while in the medium. After 195 min, a decrease in sperm-associated total acrosin (42%; p less than 0.05) was accompanied by an increase in the total acrosin level in the medium (115%; P less than 0.05). No change in the relative proacrosin content (percent of total acrosin) was evident in either medium or spermatozoa. Additional experiments quantified acrosin and proacrosin during the progression of the acrosome reaction. Both the loss of sperm-associated total acrosin and the increase in total acrosin levels in the medium were highly correlated with the fraction of acrosome-reacted spermatozoa (r = 0.954 and 0.922, respectively; P less than 0.001). However, the rate of acrosin appearance in the medium was only 60% (P less than 0.001) of the rate of acrosin loss from the spermatozoa. The fractional proacrosin content of spermatozoa (94%) and medium (31%) remained unchanged during the acrosome reaction (r = 0.15 and 0.30, respectively; P greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

Structural and functional studies on an FtsH inhibitor from Bacillus subtilis

The small 3 kDa SpoVM protein is essential for development of the spore in Bacillus subtilis. Genetic and biochemical experiments have shown that the function of SpoVM is to inhibit the proteolytic activity of FtsH during sporulation. We have used a combination of genetic and biophysical techniques to characterise the role of this small polypeptide. SpoVM was found to be widespread in Bacillus as well as in two Clostridia species, suggesting that SpoVM provides a common mechanism for inactivating the FtsH protease during spore differentiation. Using site-specific mutagenesis, we have identified C-terminal residues of SpoVM essential for biological activity. Analysis of SpoVM’s structure showed that it is able to assume an α-helical conformation in the presence of a lipid interface which may be important in interacting with FtsH.     

3-Mercaptopyruvate sulfurtransferase activity in guinea pig and rat tissues

Activities of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) in guinea pig tissues were determined and compared with those in the corresponding rat tissues. The activities in guinea pig tissues were found to be very low. The activity in the liver was 145.9 mumol pyruvate formed per g of fresh tissue per 15 min. This was 1/50 of the activity in the rat liver. Activities in the kidney and brain were 1/100 of the corresponding rat tissues. Those in the erythrocyte and heart were negligibly low and far less than 1/1000 of these tissues in the rat. Similarities between the guinea pig and patients with beta-mercaptolactate-cysteine disulfiduria are discussed.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

A dehydroepiandrosterone sulfatase inhibitory activity in soluble proteins of guinea pig liver

An inhibitor of microsomal dehydroepiandrosterone sulfatase was found in the soluble fraction of non-pregnant guinea pig liver. The extent of inhibitory effect was dependent on the concentration of soluble proteins. The inhibitor was partly purified by gel permeation and hydroxylapatite chromatography with a purification factor of 16.6. The soluble inhibitor was non-dialyzable, not destroyed by RNase or DNase digestion but totally destroyed by pronase digestion. The inhibitor is a soluble protein with a molecular weight of approximately 17,000 (determined by gel permeation chromatography). Inhibition of microsomal dehydroepiandrosterone sulfatase by the soluble inhibitor is a non-competitive inhibition. From this present finding the question arises whether the inhibitor could be involved in the regulation of the hydrolysis of dehydroepiandrosterone sulfate in the guinea pig liver.     

Androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin II, a WAP-motif protein

We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.     

Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.