Investigations on adenosine 3',5'-monophosphate phosphodiesterase in ram semen and initial characterization of a sperm-specific isoenzyme.

Phosphodiesterase is shown to occur in ram semen, and its activity to be higher in spermatozoa than in seminal plasma. Using similar substrate levels, the rate at which adenosine 3',5'-monophosphate (cyclic AMP) is metabolized by phosphodiesterase in spermatozoa is about 100 times higher than that of cyclic AMP synthesis by adenylate cyclase. In spermatozoa, phosphodiesterase is present partly in a soluble form, and partly bound; both forms can be extracted by sonication. The soluble enzyme (pH optimum 8-0, Km = 1-5 muM, mol. wt 165,000) occurs as a single isoenzyme, as shown by polyacrylamide gel electrophoresis and anion-exchange chromatography; this isoenzyme appears to be specific for spermatozoa and its formation in the testis coincides with the appearance of spermatozoa. The bound sperm enzyme has been solubilized with Trion X-100; it is a single isoenzyme (pH optimum 8-0, mol. wt 165,000) which is electrophoretically different from the soluble form, but similar to the phosphodiesterase found in other tissues. Seminal plasma phosphodiesterase (pH optimum 8-8, mol. wt 165,000) is present in the form of three isoenzymes; all three are different from the two forms of sperm phosphodiesterase, but are similar to the isoenzymes found in certain male accessory organs.     

Alkaline phosphatase isoenzymes in human kidney and urine,immunohistochemical, immunological and biochemical characterization

Summary Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%–4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%–40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.     

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.     

Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase

1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.     

Variations in the distribution of calcium, magnesium and inorganic phosphorus within chronically catheterized sheep conceptuses during the last eight weeks of pregnancy

The concentrations of calcium, magnesium and inorganic phosphorus were higher in foetal arterial plasma than in maternal jugular plasma in sheep examined between 90 and 145 days of gestation. During the same period the calcium and magnesium concentrations of foetal urine were usually less than amniotic fluid values which in turn were less than maternal plasma concentrations. In allantoic fluid, calcium concentrations were usually less and magnesium concentrations greater than maternal and foetal plasma values. A 2-5 fold increase in the calcium concentrations of allantoic fluid after superfical uterine surgery and in amniotic fluid from a group of foetuses that were exposed during operation, were considered to be artefacts of technique. Inorganic phosphorus concentrations in foetal urine, amniotic fluid and allantoic fluid were variable.     

Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

Polyacrylamide gel electrophoresis of rat brain acetylcholinesterase: isoenzymes of normal rat brain

Abstract— Triton-solubilized acetylcholinesterase (EC 3.1.1.7) of rat brain was submitted to vertical flatbed polyacrylamide gel electrophoresis. Three anodally migrating isoenzyme zones with low relative mobilities could be resolved, each of which on quantitative densitometry appeared to consist of more than one subzone. More than 50 per cent of the total AChE activity was exhibited by the isoenzyme zone closest to the origin (isoenzyme zone 3). Regional differences in AChE isoenzyme activity were quantitative only with the caudate-putamen complex, midbrain, pons and medulla oblongata exhibiting relatively high content of the three isoenzymes and the cerebral cortex and olfactory bulb possessing weak isoenzyme activities. Intermediate levels of isoenzyme activities were observed in the cerebellum and hippocampus. In all areas examined, the relative percentage values for each isoenzyme remained constant. AChE isoenzymes from the forebrain, brain stem and cerebellum of 15- and 30-day-old rats appeared to have identical patterns. In brain stem, no quantitative differences could be detected in the isoenzyme activities between 15 and 30 days of age. At both ages, the isoenzymes of male and female rats did not show any qualitative differences. The single cholinesterase (EC 3.1.1.8) isoenzyme which could be identified in brain stem supernatants of 30-day-old rats was weakly reactive and appeared to have the same relative mobility as the major acetylcholinesterase zone, zone 3. Acetylcholinesterase isoenzymes failed to demonstrate any differential response toward varying concentrations of inhibitors and to changes in pH. While there were basic similarities in the acetylcholinesterase and cholinesterase isoenzyme patterns of brain, serum, liver, skeletal muscle and intestine, brain alone exhibited a marked preponderance of the acetylcholinesterase isoenzyme zone 3.     

Amniotic fluid arginase in women at term]

Study on 34 cases. A considerable arginasic activity is observed in amniotic fluid in women at the end of pregnancy. This activity is weak, but increased by Mn2+ (about 5 to 8 times). The total amniotic fluid is more active than the surpernatant, the deposit of which has been eliminated. An important part of arginasic activity results from the elements of the deposit (amniotic cells, foetal cells and eventualy erythrocytes in hemorragic amniotic fluid).     

Characterization of proteolytic activities in embryos of Xenopus laevis

1. Proteolytic activities in early embryos of Xenopus laevis exhibited maximum levels at pH 3.2, 5.6 and 7.2 when 3H-BSA was used as substrate, and the maximum proteolytic activity at pH 3.2 was several thousand-fold higher during the tail bud stage than in the unfertilized egg. 2. The proteolytic activity at pH 3.2 was separated into two fractions by gel chromatography. One fraction corresponded to a mol. wt of about 40,000 and its activity was inhibited by thiol protease inhibitors. The other appeared to be a protease of much higher mol. wt. 3. The maximum activities at pH 5.6 and 7.2 appear to correspond to proteins of mol. wt greater than 1,000,000.     

Effect of Heat Treatment on the Development of Polygalacturonase Activity in Tomato Fruit during Ripening

When mature green tomato fruits are stored at 22?C for 30 days,they ripen normally and soften, but if they are kept at 33?Cfor 15 days (heat treatment), then stored at 22?C they do notsoften. The effect of heat treatment on the development of polygalacturonase(PG, EC 3.2.1.15 [EC] ) activities in tomato fruits during storagetherefore was studied. When mature green tomato fruits werestored at 22?C, PG activity, which had not been detectable inthe fruits, appeared as the color changed and increased dramaticallythereafter. PG activity, however, did not appear during heattreatment. When heat-treated fruits were transferred to 22?C,PG activity appeared after a 6-day lag period and increasedduring the next 30 days at 22?C to about 15% of the value detectedin ripe tomato fruits. The PG in ripe tomato fruits was composed of two isoenzymesthat had different mol wts. A high molecular form (PG-1, molwt 100K) appeared during the early stage of ripening and a lowmolecular form (PG-2, mol wt 44K) a little later. PG-2 increasedvigorously during ripening and eventually accounted for mostof the enzyme activity in the ripe fruits. Only a single isoenzyme(Y-PG, mol wt 100K), however, was detected in heat-treated tomatofruits stored at 22?C for 30 days. PG-1 and Y-PG gave the samemol wt on Sephacryl S-200 gel nitration, but could be separatedby Toyopearl HW-55 F gel filtration. (Received October 31, 1983; Accepted February 20, 1984)     

A comparative study on the peroxidase activity within the aqueous humor of the rabbit, guinea-pig and rat

Aqueous humor of the rabbit, guinea-pig and rat has been examined to determine if non-specific peroxidase activity is present within this ocular fluid. Substantial peroxidase activity was found within the aqueous humor of the rat. In contrast, no peroxidase activity could be detected in the aqueous humor of the rabbit or guinea-pig. Partial purification by gel chromatography revealed that the peroxidase within the aqueous humor of the rat has a mol. wt of approx. 45,000.     

The application of QAE-Sephadex for the purification of two staphylococcal enterotoxins. I. Purification of enterotoxin C2.

A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Effects of thrombin on washed, human platelets: changes in the subcellular fractions.

Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecysulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 plus or minus 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control (mol. wt 147 000 plus or minus 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37 degrees C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 plus or minus 3500) had disappeared. The distribution of protein and beta-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (greater than 77%). The granule fractions, D and E of the control contained 3.0% plus or minus 0.8% and 6.4% plus or minus 1.3% of the total amount of beta-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% plus or minus 1.0% of total while fraction D was lacking.     

Cellular distribution of elongation factor EF1 in wheat germ]

Cellular distribution of elongation factors (EF1) from imbibed then redessicated wheat embryos is determined after purification and analytical gel electrophoresis of soluble and ribosome-bound factors. Two heavy forms (EF1 H, mol. wt, 250 000) are found in cytosol while ribosome-bound factors contain a light form (EF1L, mol. wt, 45 000) with the greatest activity and a heavy form (mol. wt, 160 000) which might well be an intermediary in the recycling of ribosomal factor EF1L to soluble factor EF1H.     

Gestational profiles of rat placental lactogen-II (rPL-II) and growth hormone (GH) in maternal and fetal serum, amniotic fluid, and placental tissue.

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

An irreversible tissue inhibitor of collagenase in human amniotic fluid: Characterization and separation from fibronectin

Soluble fibronectin isolated from human plasma and amniotic fluid by gelatin-Sepharose affinity chromatography was tested for inhibitory activity against specific collagenase secreted by human and rabbit fibroblasts. The fibronectin preparation derived from plasma showed little inhibition, but the one derived from amniotic fluid contained potent inhibitory activity against collagenase. This activity was separated from fibronectin on a DE-52 cellulose column and did not cross-react with antibodies to fibronectin. The inhitor was a glycoprotein that was partially purified from amniotic fluid by concanavalin A-Sepharose affinity chromatography. Inhibition was irreversible and enzyme activity was not recovered after reaction with latent or activated collagenase by either trypsin or organomercurial treatment.     

Comparative effects of soil organic matter fractions on phosphatase activities in wheat roots

Summary Soil organic matter was obtained from two agricultural soils using alkali extraction followed by acidification to produce humic and fulvic acids which were further fractionated by adsorption and gel chromatography.All the products inhibited the activity of phosphatase prepared from wheat roots, but to different extents. Humic acids produced a greater inhibition of enzyme activity than either the fulvic acids or water extracts of soil. Aspergillin, fromAspergillus niger, had a similar C, H and N content to humic acid and produced a similar inhibition of phosphatase activity.The inhibitions produced by corresponding fractions derived from the two soils were slightly different, but the trends between similar fractions from different soils were comparable. The lower mol. wt. components of humic acid inhibited phosphatase activity to a greater extent than higher mot. wt. fractions. Although fulvic acid comprised only low mol. wt. components it was less effective in inhibiting enzyme activity than those components of comparable mol. wt. present in the corresponding humic acid. Synthetic polymaleic acid, produced an inhibition of phosphatase activity similar to that caused by fulvic acid.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.