Estrone concentration in the peripheral plasma of pregnant and non-pregnant gilts

A rapid radioimmunoassay for estrone (total unconjugated and sulfated) was developed to determine plasma estrone (E1) concentrations in inseminated gilts that conceived and those that had not. Thirty-one 160 day-old prepuberal gilts were induced to ovulate with gonadotropins and were artificially inseminated 10 hr before the expected time of ovulation (Day 1 - day of insemination). Unconjugated E1 and E1SO4 were extracted from 20 to 500 microl of plasma twice with 5 ml of tetrahydrafuran:ethyl acetate (1:1). Aliquots of a standard E1SO4 preparation were dissolved in 500 microl of distilled water and extracted at the same time as the plasma samples. The dried extracts were solvolyzed for 1 hr at 50 degrees C in 0.6 ml of glacial acetic acid:ethyl acetate (1:1), and the dried residue was redissolved in 0.2 ml of distilled water and extracted once with 2 ml of diethyl ether. Twenty of 31 gilts were pregnant at Days 29 to 31 of the induced cycle. Plasma E1 in pregnant gilts increased from 85 pg/ml on Day 18 to 702, 1879 and 2793 pg/ml, respectively, on Days 22, 25 and 29 to 31. Three of the non-pregnant gilts had plasma progesterone secretion maintained until Day 22; they also had a transitory increase in plasma E1 on Day 22 (215 pg/ml). Some blastocysts may have been present to exert a temporary luteotropic effect, but not enough blastocysts to completely overcome the luteolytic effect of the uterus. Quantification of plasma E1SO4 could be used as a pregnancy test in the pig.     

Immunoglobulin levels,protein concentrations and alkaline phosphatase activity in uterine flushings from mares with endometritis

Uterine samples and flushings were obtained from 87 mares to compare endometrial bacteriology and biopsy with immunoglobulin and protein concentration and alkaline phosphatase activity in uterine flushings. Mares were designated as infected if both bacteriology and biopsies were positive. The immunoglobulin levels, protein concentration and alkaline phosphatase activity in uterine flushings from infected and non-infected mares were compared. Twenty (23%) of the mares were classified as infected. A significantly higher proportion of infected mares (cf. non-infected) had elevated IgA and protein concentrations. Levels of IgG, IgG_T or alkaline phosphatase were not significantly elevated in infected mares. These results suggest that IgA and protein levels are elevated in the uterus in the presence of active infection.     

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.     

The haemocytes and the activities of leucine aminopeptidase and alkaline phosphatase during development of Ceratitis capitata: Specific secretion of a leucine aminopeptidase isozyme

• 1.1. The types of haemocytes during larval development were studied.
• 2.2. The developmental profile of leucine aminopeptidase and alkaline phosphatase was studied. The maximum LAP activity was found to be in early larval development, while the maximum alkaline phosphatase during the white pupal stage.
• 3.3. These activities were compared with those determined in cell-free haemolymph.
• 4.4. Both hydrolytic enzymes have been found histochemically in the prohaemocytes and in the plasmatocytes.
• 5.5. In cultured haemocytes experiments it was found that 64% of the total LAP activity was secreted into the incubation medium, while electrophoretic analysis of released LAP activity demonstrated that only LAP A isozyme was secreted.
• 6.6. Based on the above results we suggest that both hydrolytic enzymes are functionally important throughout larval development.

     

Purification and partial characterization of aminopeptidase A from the serum of pregnant and non-pregnant women

Aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7) was purified from human maternal and control sera using CM-cellulose chromatography, DEAE-Sephacel chromatography, Sephacryl S-300 gel filtration and hydroxyapatite chromatography. The purification coefficients were 3069 and 5210 and the yields 6.3 and 6.1% for the maternal and control serum, respectively. The purified enzymes appeared free from other serum aminopeptidases in polyacrylamide gel electrophoresis. The biochemical and physical characteristics of the enzymes from maternal and control sera were similar. A molecular weight of 260 000, an optimum at pH 6.75-7.25 and a fairly good stability of the enzymes at 4 and -18 degrees C were recorded. The alkaline earth metals (Ca2+, Ba2+, Sr2+) were the activators of alpha-L-glutamyl-beta-naphthylamide hydrolysis, while alpha-L-aspartyl-beta-naphthylamide hydrolysis was markedly potentiated with Ca2+ but not with Ba2+ at all. The most rapid hydrolysis was shown with GluNA (Km with Ba2+ 0.156 +/- 0.014 mM and 0.136 +/- 0.009 mM in maternal and control serum, respectively), while only minimal hydrolysis of some neutral and basic amino-acid-beta-naphthylamides were observed. The contribution of the placenta to the elevated aminopeptidase A levels in the pregnancy plasma could not be solved on the basis of the present observations.     

Preparation and P content of non-pregnant and pregnant human uterine myosin

Myosin content and phosphorus (P) concentration of myosin preparations were measured in non-pregnant and pregnant human myometrial tissue specimens. It was found that the amount of myosin gained from 1 g of minced myometrial tissue is 0.5 mg in the early follicular phase of the menstrual cycle, 0.6-0.7 mg in the late luteal phase, and 6-7 mg during pregnancy. Considering the different functional stages of the myosin sources and the performance characteristics of the methods, the estimated myosin content of non-pregnant myometrium is 1.0-1.5 mg, while 10-15 mg in pregnant myometrial tissue. A considerable amount of P is bound to the preparations. It is the smallest in the post-menstrual period and increases towards the end of the cycle. The largest amount of P is gained from fresh pregnant uterine samples. Analysis of the alkaline hydrolysate showed that the phosphate group was bound to amino acids, in the largest amount to arginine, less to histidine and the smallest amount to lysine and serine. As a function of the duration of storage, especially the P-Arg concentration was decreasing. The prolonged hydrolysis time decreases again the concentration of P-Arg with a consecutive increase of No. 1 and 2 P-containing peaks in the chromatographic profile of alkaline hydrolysate.     

Intestinal leucine aminopeptidase and alkaline phosphatase: Genetic regulation and development in mice

Intestinal and serum leucine aminopeptidase (LAP) and alkaline phosphatase (AKP) were characterized by electrophoresis for eight inbred strains of laboratory mice. Intestinal LAP and AKP of adult mice were expressed concordantly within strains, as banded or diffuse, and concordantly for rate of migration within strains that had diffuse isozymes. All strains, except DD/S, had a single band of serum LAP and a single, diffuse zone of serum AKP. DD/S had a double band of serum LAP as well as isozymes of intestinal LAP and AKP unlike those of other strains. All strains displayed similar, neuraminidase-sensitive isozymes of intestinal LAP and of AKP prior to weaning, but after weaning there was marked sensitivity to neuraminidase only in DD/S. In interstrain crosses, banded/diffuse, migration rate, and neuraminidase sensitivity were inherited as independent autosomal traits, with indications of variable penetrance and genetic interaction. Support was provided by NIH Grant RR08117.     

Activity of the gamma-glutamyltransferase, leucine aminopeptidase and alkaline phosphatase enzymes in human tear fluid

In this work the presence of gamma-glutamyltransferase (GGT), leucine amino-peptidase (LAP) and alkaline phosphatase (AP) is shown in human tear fluid. We studied these levels according to sex, age and some eye refraction defects. The differences between the levels for both sexes are not significant. LAP and AP do not show any differences in either age groups or individuals with some refraction defects. The average level of GGT is higher from 40 years of age upwards (p less than 0.005). In individuals with refraction defects, the enzymatic activity is significantly higher (p less than 0.05) than the activities found in normal subjects. The levels of the three enzymes in serum and tear fluid do not show a significant correlation nor are they significantly modified after the samples have been frozen for a month at -20 degrees C.     

Estimates of leucine aminopeptidase activity in different marine and brackish environments

AIM: Leucine aminopeptidase (LAP), an enzyme involved in the decomposition of natural peptides, was measured in different marine and brackish ecosystems, together with some environmental and microbiological parameters. METHODS AND RESULTS: The fluorogenic compound L-leucine-7-amido-4-methyl coumarin was specifically used for the determination of this in situ activity. The enzyme data obtained from this comparative study highlighted the strong spatial and temporal variability of the distribution of LAP in aquatic ecosystems, which was sometimes related to the course of environmental variables such as salinity and organic carbon content. CONCLUSIONS: LAP assay has proved to be a rapid method providing useful information on the microbial metabolic processes involved in the mineralization of organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The determination of the potential rates of extracellular enzyme activity is of great ecological importance to extend knowledge on the role played by bacteria in aquatic biogeochemical cycles.     

Characterization of a secretase activity for placental leucine aminopeptidase

Placental leucine aminopeptidase (P-LAP) is believed to play an important role in the inactivation of small regulatory peptides. P-LAP exists in both membrane-bound and soluble forms and cDNA cloning has demonstrated that P-LAP is a type II membrane protein, which means that its soluble form is released by a specific proteolytic cleavage. In this report, we studied this process in COS7 cells. Inhibitors of serine or aspartic proteases did not affect the secretion of P-LAP, while EDTA and 1,10-phenanthroline inhibited it. In addition, we transfected P-LAP expression vectors that have point mutations of the cleavage site or deletion of the juxtamembrane stalk. Point mutations of the cleavage site resulted in significantly lower secretion of P-LAP. On the contrary, the distance to cleavage site showed no relation to P-LAP secretion. These results suggest that P-LAP secretase has a metalloprotease activity which depends on the amino acid sequence of the cleavage site.     

Acid phosphatase activity in human uterine fluid in relation to infertility.

In the luminal fluid of parous women the acid phosphatase activity was found to be very low and remained virtually unchanged throughout the cycle (10.60 to 18.14 mgp/hr/100 ml). In the infertiles, the activity was generally high with a maximum of 54.37 mgp/hr/100 ml at late follicular phase. It was argued that increased enzyme level in infertiles could have adversely affected the sperm capacitation power as well as the implantation of the fertilized ovum.     

Collagenase activity in cervical tissue of the non-pregnant and pregnant human cervix

Collagen degradation has been suggested to play an important role in the process of cervical ripening at term. After giving informed consent tissue samples were taken from the posterior lip of 10 non-pregnant women of child-bearing age, 10 women having a termination of pregnancy at 9-12 weeks' gestation and 12 women who had had normal spontaneous deliveries at term. Collagenase activity was measured by a specific technique using native, triple helical collagen. Proteolytic activity was determined with commercially available 14C-methaemoglobin as substrate. For identification of collagen fragments SDS-polyacrylamid-gel-electrophoresis was performed using a modified Laemmli-technique. Collagenase and protease activity were found in all extracts of the different groups. There was a marked increase in enzymatic activities in the postpartum tissue samples suggesting that significant collagen breakdown had taken place. The absence of typical collagen fragments in the SDS-electrophoresis seemed to be due to a very rapid further degradation of the fragments by proteases into small peptides which then readily left the cervix. The present findings confirm the essential role of proteolytic enzymes in cervical dilatation at term.     

An investigation of the uterine luminal environment of non-pregnant and pregnant pony mares.

Uterine flushings were collected from 30 non-pregnant Pony mares on Days 8, 12, 14, 16, 18 or 20 after oculation. Mares were allowed a recovery period of one oestrous cycle and were mated at the next oestrus. They were then ovario-hysterectomized on days which corresponded to the day of the oestrous cycle to which they were assigned. Uterine flushings were analysed for total recoverable protein and acid phosphatase activity. Least squares analysis indicated a status X day interaction for total protein (P less than 0.10) and acid phosphatase activity (P less than 0.005) in which the latter was higher in uterine flushings during pregnancy. Peripheral plasma oestrone and oestradiol concentrations were measured by radioimmunoassay and results indicated that plasma oestrone concentrations in pregnant and non-pregnant mares were not different, and oestradiol was lower (P less then 0.005) in the peripheral plasma during pregnancy. conceptus membranes were incubated in vitro for 120 min in a chemically defined medium. Incubation medium was then assayed to assess oestrone and oestradiol production capacilities at Days 8, 12, 14, 16, 18 and 20 of pregnancy. Conceptus membrane production of oestradios (pg/5 ml/h) increased (P less than 0.05) from Day 8 (243 pg/5 ml) to Day 20 (108 763 pg/5 ml). A similar trend, but of lower magnitude, existed for oestrone production.     

Activity of alkaline phosphatase in uterine flushings of dairy cows affected with ovarian cysts or endometritis

Both uterine horns of 14 dairy cows with ovarian follicular cysts, and four animals affected with purulent endometritis were flushed via catheter using 30 ml phosphate buffered saline, following evisceration at a local abattori. Activity in the flushing media of alkaline phosphatase (ALP) and aspartate aminotransferase (GOT) were examined. Ovaries were prepared for light microscopy. Amount and morphological integrity of luteinized tissue found on the ovaries were reflected by correspondent levels in ALP activity, which was higher in the media taken from the ipsilateral to the luteal tissue situated uterine horns (651 +/- 228 vs 244 +/- 62 u/l, n = 3). Only cows having relatively large amounts of luteal tissue on the cystic ovaries (as in luteinized follicular cysts) exhibited very high ALP activity in uterine flushings (2693 +/- 1348 u/l, n = 2). Results suggest the existence of local relationships between luteal tissue in the ovary and the ipsilateral uterine horn in cows with ovarian follicular cysts.     

Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.