Insulin-like growth factor binding proteins and mitogenic activity of partially fractionated sheep amniotic fluid.

Amniotic fluid collected from ewes on various days of gestation was examined for the presence of insulin-like growth factor (IGF) binding proteins. IGF-binding proteins with a molecular mass of 40-45 kDa appeared at day 41 of gestation. The level of these major IGF-binding proteins increased during pregnancy and reached a maximum at day 106. Smaller IGF-binding molecules with an approximate molecular mass of 35 kDa and 25 kDa appeared at day 90, also reaching a concentration peak at day 106. The mitogenic activity of sheep amniotic fluid after chromatography on Sephadex G-50 was separated into two peaks. The peak having lower molecular mass corresponded to an elution profile of 125I-IGF-I. The first peak, having higher molecular mass, was eluted immediately after the void volume of column. Electrophoresis and ligand blotting showed that proteins in the first peak had similar properties as IGF-binding proteins.

Mitogenic activity of high molecular weight forms of insulin-like growth factor-II in amniotic fluid.

Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

Insulin-like growth factor binding proteins and their functions (minireview).

Insulin-like growth factor (IGF) action is influenced by the insulin-like growth factor binding proteins (IGFBPs). Since 1988 eight forms of IGFBPs have been found which differ in molecular weight, amino acid composition, distribution in biological fluids and influence upon IGF activity. An important biological property of the IGFBPs is their ability to increase the halflife of the IGFs in the blood. They are able to act as potentiators of IGFs activity on the cell proliferation. As IGFBPs bind to cell surfaces, they may act either to deliver the IGFs to those surfaces either for the activation of specific receptors or cell responses independently of receptor activation. Posttranslation modification such as phosphorylation, glycosylation and proteolysis of IGFBPs influence their affinity to IGFs. In addition, the IGFBPs may also act as inhibitors to block the activity of the IGFs by preventing cellsurface binding.

[Peptide-like substances in sheep amniotic fluid which regulate proliferation of BP-A31 cells]. / Peptidické látky amniónovej tekutiny oviec regulujú proliferáciu buniek BP-A31.

Cell proliferation and differentiation of developing fetus is influenced by hormones as well as insulin-like growth factors and their binding proteins contained in amniotic fluid. Our purpose was to study the actual mitogenic activity of proteins and peptides present in the sheep amniotic fluid. The cell cycle regulatory activity was estimated by using mouse fibroblasts BP-A31 as target cells. The whole amniotic fluid was inactive. However, after removal of small molecules on Sephadex G-10, the fraction eluted in the void volume (M(r) > or = 0.7 kDa) was able to induce the cell division cycle in a significant proportion of quiescent fibroblasts (Fig. 1, fraction A; Fig. 2). By further gel chromatography of this active fraction at acidic condition on Sephadex G-50, two components with mitogenic activity were separated. One component was eluted immediately after the void volume of the column, the other one was coeluted with 125I-IGF-I (Fig. 3). The functional characteristics of mitogenic signal of both components (sensitivity to mitogenic effectors) were similar to those of IGF-I and insulin (Fig. 4). We suppose that a component with higher molecular weight eluted in the vicinity of the void volume of Sephadex G-50 represents probably IGFBPs or other similar proteins.

Stress relaxation in cherry fruit.

Stress relaxation in three varieties of cherries was studied using the penetration test with a cylindrical pin, 4 mm in diameter, having a flat tip. One half of the fruit was modified by partial skinning, since experiments show that skin approximately doubles the apparent modulus of elasticity of the cherries. The skin also obscures a part of the fruit's distinctiveness, and affects the experimental results, even if a part of the fruit has been removed. A thermal activation theory with the residual part of stress was used to evaluate the stress relaxation experiments, and yielded results very similar to those previously obtained for vegetable flesh. Real consistency was observed mainly for residual stress and activation volume. It was observed that the relation between parameters Ct and log bt, and the initial relative slope of the relaxation curve provides information on fruit firmness.

Indirect examination of exocrine pancreatic function in vivo by determination of chymotrypsin activity in animal intestine using a synthetic substrate.

Using the substrate N-acetyl-L-tyrosyl-p-aminobenzoic acid, we determined chymotrypsin activity in the small intestine of calf, pig, and poultry. Orally administered N-acetyl-L-tyrosyl-p-aminobenzoic acid is enzymatically cleaved in vivo, and the released p-aminobenzoic acid is determined by HPLC. We found that the p-aminobenzoic acid concentration in plasma and urine was significantly influenced by the feeding of soya flour. After soybean flour feeding, the p-aminobenzoic acid concentration significantly increased in the plasma of calves and hens, in contrast to pigs, where the p-aminobenzoic acid concentration significantly decreased. This shows that the oral administration of N-acetyl-L-tyrosyl-p-aminobenzoic acid with subsequent determination of p-aminobenzoic acid is suitable for the estimation of exocrine pancreatic function and for determination of changes in intestinal proteolytic activity caused by antinutritive substances.

Lactate dehydrogenase isoenzyme patterns in bird, carp and mammalian sera.

Isoenzyme patterns of lactate dehydrogenase (EC 1.1.1.27) in avian sera are described and compared with those of carp and some mammals. The predominant portion of lactate dehydrogenase in avian sera was concentrated in the muscular form of the enzyme (lactate dehydrogenase 5). Most mammalian sera (with the exception of rat serum) showed a different pattern, in which the main portion of lactate dehydrogenase activity migrated in the first three anodic fractions. Fish serum lactate dehydrogenase isoenzymes were distributed similarly to those of mammals. The electrophoretic mobility of bird lactate dehydrogenase isoenzymes in a pH gradient of 3 to 9 was different from that of carp, cattle and rabbit. Bird lactate dehydrogenase isoenzymes were localized in the pH region of 5.8 to 8.1. In contrast, the mammalian isoenzymes were more acidic, with pI values in the range of 4.5 to 7.3. Lactate dehydrogenase isoenzymes of carp migrated in a narrow pH range of 5.2 to 6.5.

Simultaneous determination of p-aminobenzoic acid and its metabolites in urine by high performance liquid chromatography.

We developed a new HPLC method for the determination of p-aminobenzoic acid (PABA) and its metabolites (p-aminohippuric acid, N-acetyl-p-aminohippuric acid, N-acetyl-p-aminobenzoic acid) in urine. As the internal standard m-hydroxybenzoic acid was used. In the isocratic elution the mobile phase consisted of methanol and 0.02 M ammonium acetate (20:80 v/v, pH 4.0). The separation was carried out on the C18, reversed-phase column, particle size 5 microns. The separated components were detected at 280 nm. The method can be used in the assessment of the response of pancrease (secretion of digestive enzymes) to soya feeding as well as in the diagnosis of the exocrine pancreatic diseases of animals.

[New findings on serum lactate dehydrogenase isoenzymes in pigs during slaughter]. / Nové poznatky o izoenzýmoch laktátdehydrogenázy v krvných sérach osípaných pri porázke.

In the article we describe lactate dehydrogenase-(LD) (EC 1.1.1.27) isoenzyme pattern detected in the sera of pigs at slaughter. The pattern was different from that of normal serum (Fig. 1) and was characterized by the occurrence of an extra LD-fraction in the cathodic site of LD4 (Fig. 2). This fraction was unusual due to its unwillingness to separate by native polyacrylamide gel electrophoresis (PAGE) and took shape of a diffuse zone. The presence of the extra LD-zone caused a proportional decrease in quantitative distribution of the other LD forms, especially LD1 to LD3, in slaughtered pig sera (Tab. I). We examined the homogeneity of an apparent LD5-fraction using gel isoelectric focusing (IEF). We found out that after separation in a gradient of pH (3-9) two to three new extra bands with LD activity appeared in the area with relatively high pH value (pH 9) (Fig. 3). Their localization in the gradient of pH was greatly different from that of true LD molecules, the latter being situated in more acidic area. It is obvious from the finding described above that the diffuse LD-zone, detected in the serum of pigs at slaughter by native PAGE, was in no case a homogeneous protein. Consequently, it eliminates a possibility that the extra LD fraction reflects an increased LD5 activity in serum of affected animals. On the contrary, the IEF showed that the diffuse LD-zone consisted of two to three electrophoretically distinct proteins with relatively high pI values. As these proteins differed in their electrophoretic properties from the true LD isoenzymes we denoted them LD-like proteins. An origin of the unusual LD-like proteins detected in the serum of pigs at slaughter remains unknown for us for the time being.

[Lactate dehydrogenase isoenzymes in mammalian and chicken serum]. / Izoenzýmy laktátdehydrogenázy v cicavcích a kuracích sérach.

In the article we describe characteristics of lactate dehydrogenase (LD) (EC 1.1.1.27) isoenzymes of fowl origin. We compare them with characteristics of evolutionary different mammalian forms of the enzyme. Separations of LD isoenzymes have been done using the most progressive electrophoretic techniques available at present. They included electrophoresis in homogeneous and gradient polyacrylamide gels (PAGE) as well as isoelectric focusing (IEF). A typical densitometric pattern of serum LD isoenzymes obtained by gradient PAGE is illustrated in Fig. 1. It is obvious that LD isoenzymes of all investigated animals have been well separated under applied experimental conditions, with an exception of fowl serum. We succeeded in separating fowl LD isoenzymes using isoelectric focusing. It was possible to distinguish five fractions of lactate dehydrogenase (Fig. 2) by this technique. As shown in Fig. 2, mobility of avian isoenzymes in the gradient of pH differs from that of their mammalian analogues. Fowl LD isoenzymes were localized in cathodic part of IEF-gram. In the case of mammalian isoenzymes (cattle and rabbit), we found LD4 and LD5 forms of the enzyme in this part. The major fractions, i.e. LD1 to LD3 were present in anodic part of IEF-gram. We quantitatively expressed this different mobility of mammalian and avian forms of LD using retention factors Rf (Tab. III). A migration distance of cattle LD1 served as a reference point. It could be seen from a comparison of Rf values that avian forms of LD (of galiform origin) differed from mammalian ones by an outstanding shift of their isoelectric points, especially that of LD1, toward basic values. The total LD activity (IU) and the relative distribution of the LD isoenzyme activities (%) in normal serum of various animals are listed in Tabs I and II. A comparison of these values showed that, in contrast to mammalian serum (with the exception of the rat one), LD5 was the predominant fraction in fowl serum, followed by LD4. LD1 to LD3 occurred in low amounts with fairly similar proportions. On the other hand, most of mammalian serum revealed a reverse pattern of LD isoenzymes, i.e. predominant portion of serum activity had been concentrated in the first three anodic fractions and LD4 and LD5 had been found to be minor ones.

[Determination of the enzymatic activity of trypsin and chymotrypsin using denatured 125I-labeled albumin]. / Stanovenie enzymatickej aktivity trypsínu a chymotrypsínu pouzitím denaturovaného 125J znaceného albumínu.

The method of the determination of trypsin and chymotrypsin activity by means of modified 125I-labelled albumin is described. The radioalbumin, denatured with an alkaline solution of urea, is about 20 times more sensitive to enzymatic hydrolysis than native radioalbumin. Owing to the higher sensitivity of the proposed procedure, as compared with other available techniques, this method enables accurate determination of lower enzyme concentrations than it has been possible until now. The method is suitable for the measurement of the rate of the proteolysis of raw enzymatic extracts as well as for exact kinetic measurements of purified enzymes.

[Distribution of acid and neutral proteases in various organs and tissues of chickens]. / Distribúcia kyslých a neutrálnych proteináz v niektorých orgánoch a tkanivách kurciat

The radioisotopic method of 131J-labelled albumin was employed to determine the distribution of acidic proteinase activity in some organs and tissues of chickens. The highest enzymatic activities were found in intestine wall, in pancreas, and in liver. Considerably lower activities were ascertained in kidneys, brain, lungs, and heart. The different proportions of these enzymes in homogenates and supernatant fractions (106 000 g) testify to a lack of uniformity in the solubility of cathepsins in the organs tested. The tested organs, with the exception of pancreas, did not show any enzymatic activity of neutral proteinases.

[Electrophoretic characteristics of arylamidases in some organs of cattle]. / Elektroforeticke vlastnosti arylamidáz z niektorých orgánov hovädzieho dobytka

Agar electrophoresis was employed to study the homogeneity of arylamidase enzymes in the soluble fractions of liver, kidney, heart, spleen, brain, pancreas, and muscle homogenates and in serum. Enzymatic activity was determined in relation to the L-leucyl- and L-lysyl-p-nitroanilide chromogenic substrates. In all cases, electrophoresis made it possible to find at least two active zones with different electrophoretic mobility and with different activity in relation to the two substrates used.

[Electrophoretic properties of arylamidases from various cattle organs]. / Elektroforetické vlastnosti arylamidáz z niektorých orgánov hovädzieho dobytka

Agar electrophoresis was employed to study the homogeneity of arylamidase enzymes in the soluble fractions of liver, kidney, heart, spleen, brain, pancreas, and muscle homogenates and in serum. Enzymatic activity was determined in relation to the L-leucyl- and L-lysyl-p-nitroanilide chromogenic substrates. In all cases, electrophoresis made it possible to find at least two active zones with different electrophoretic mobility and with different activity in relation to the two substrates used.

[Determination of the proteolytic activity of beef liver by means of natural substrates labed with 125 I]. / Stanovenie proteolytickej aktivity hovädzej pecene pomocou prirodzených substratov znackovaných 125 J

There is a description of the determination of the enzymatic activity of acid proteinases: the method is based on the use of 125J-labelled natural protein substrates. Labelled albumin 125J, globulin 125J, and insulin 125J were tested for the determination of activities. All the substrates were hydrolyzed with the enzymes of the supernatant fraction (106 000 g) of beff liver homogenate in the zone of acid pH. Optimum comditions of enzymatic reaction were tested, the dependence of reaction on the concentration of the enzyme, on time, and on temperature was determined, pH optimum was ascertained for individual substrates, and pH stability was determined. It follows from the results that the method is suitable for the determination of the enzymatic activity of proteinases of the cathepsin character.