Purification and partial characterisation of α2-antiplasmin and plasmin(ogen) from ostrich plasma

This study reports the isolation and partial characterisation of the ostrich serpin, α₂AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α₂AP was purified using lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a M_r of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α₂AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α₂AP, DFP and EACA. Ostrich plasminogen was highly purified after lysine–Sepharose chromatography and showed a M_r of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M_r of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively.     

Prolactin and Somatotropin Binding by Granulosa Cells of Cows at Their Different Reproductive States

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3–5 to 6–10 mm, specific binding of ¹²⁵I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data indicate that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1–2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

New fluorogenic peptide substrates for plasmin

Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The Km values estimated from Line-weaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10(-4) M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, alpha-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond. These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys- of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.     

Conformation of sturgeon pituitary somatotropin

Pituitary somatotropin (growth hormone) from the sturgeon (Acipenser gulden-stadti) has been studied by zero-order and second-order absorption spectroscopy, as well as by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 15,000 +/- 110 M-1 cm-1 at 278.5 nm. Comparison of the conformations of sturgeon somatotropin and somatotropins isolated from several mammalian species, including bovine and human, indicates a close relationship between these molecules. Such similarities may be related to the relatively high biopotency of this fish hormone in mammalian assay systems.     

Observations of Lophelia pertusa and the surficial geology at a deep-water site in the northeastern Gulf of Mexico

Authigenic carbonate, precipitated in conjunction with biogeochemical activity associated with hydrocarbon and related fluid seepage, provides exposed and buried hard substrate on the crest and flanks of a low-relief mound located on the upper De Soto Slope in the northeastern Gulf of Mexico. Lophelia pertusa has successfully colonized some of this carbonate material. Individual colonies range in size from a few centimeters to over 1.5 m in diameter while aggregations of closely associated colonies with linear orientations were observed to attain 1.5–2 m in height and width and 3–4 m in length. Many of the aggregated colonies appear to be in the first phase of the `thicket' building stage described by Squires (1964). Colonies less than 50–75 cm in diameter were nearly always completely pure white. Larger colonies and the aggregated colonies are often light to dark brown in coloration at their base and center with many having only white terminal branches and some with no white corallum at all.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

Immunological and receptor-binding properties of fragments resulting from cyanogen bromide cleavage of bovine growth hormone

Fragments obtained from bovine growth hormone (somatotropin) by cyanogen bromide cleavage were isolated and identified. Their activities were investigated in a radioimmunoassay for bovine growth hormone and in a radioreceptor assay for growth hormone which uses membrane-associated receptors from the liver of a pregnant rabbit. At least one antigenic determinant and the receptor-binding site could be located in the sequence comprising residues 1-124/6-124 plus 150–179 (disulfide-linked), although they appeared not to be identical. An apparent increase in affinity compared with unfractionated cyanogen bromide-cleaved hormone was observed in both assays for the fraction containing these fragments. Neither intactness of methionyl residues nor that of the hormone appeared to be absolutely required for antibody-binding and receptor-binding activity (although other antigenic determinants may have been lost as a result of cleavage). However, the activities of the disulfide-linked fragment were low, indicating that conformational or other changes had modified the antibody-binding and receptor-binding sites.     

Human pituitary growth hormone. 44. Effects of plasmin-modified hormone and its fragments on ornithine decarboxylase activity and lipolysis.

The rat hepatic ornithine decarboxylase stimulating activity of plasmin-modified human growth hormone and its two peptide fragments has been investigated. The activity was completely retained after plasmin treatment. The NH2-terminal fragment [Cys (Cam)53-HGH-(1-134)] retained 10% of the activity, whereas the COOH-terminal fragment [Cys (Cam) 165, 182, 189-(141-191)] was not active. The lipolytic activity of human growth hormone was greatly reduced after plasmin treatment, as examined in isolated rabbit adipocytes. It is suggested that the structural requirements for the lipolytic activity of the hormone are different from those required for stimulation of ornithine decarboxylase activity.     

The role of a contaminant in thrombin in the human plasmin assay system

Many of the anomalous results obtained in the fibrinolytic assay of human plasmin systems were shown to be simply explained if bovine plasminogen had been introduced into the assay system on the addition of thrombin. Experimental investigation of the proteolytic and fibrinolytic activity of systems containing plasmin and thrombin showed that enzyme activity was influenced by the presence and quantity of thrombin. The quantity of bovine plasminogen present as a contaminant in bovine fibrinogen was shown to be responsible for only 1/25th of the observed enhanced activity. Thrombin in the amounts commonly used for clotting contained sufficient proenzyme to account for all this activity. A highly purified thrombin preparation obtained from another laboratory, and thrombin purified in this laboratory by starch electrophoresis brought about no enhancement of activity. The material separated from thrombin by starch electrophoresis was shown to be enzymatically identical with bovine plasminogen and, on labelling with radioactive iodine, was shown to behave physically like bovine plasminogen. Several experiments reported in the literature were reinterpreted in the light of this observation.     

The histochemical pattern of mechanically or chemically injured rabbit cornea after aprotinin treatment: relationships with the plasmin concentration of the tear fluid

Summary Plasmin, a serine protease, was recently found to be involved in corneal ulcerative processes in humans and rabbits. In our experiments, plasmin activity was found in the tear fluid after mechanical and chemical damage of the rabbit cornea, such as de-epithelization and burning with alkali. The plasmin concentrations in the tear fluid were dependent on the severity of injury. The highest plasmin activity (2.0–3.0 g ml^–1) occurred after severe alkali damage to large areas of the cornea, and the lowest activity (0.4–1.0 g ml^–1) after mechanical injury (de-epithelization).Plasmin concentrations up to 1.0 ml^–1 were associated with increased activities of lysosomal hydrolases in epithelial cells and keratocytes beneath the epithelium. Plasmin activities increased as the inflammatory reaction developed. When plasmin activity in the tear fluid was higher than 1.0 g ml^–1, inflammatory cells were found in the corneal stroma. Levels of 1.5–2.0 g ml^–1 were connected with higher numbers of inflammatory cells (particularly polymorphonuclear leukocytes) with increased activities of lysosomal hydrolases. Very high plasmin activities (2.5–3.0 g ml^–1) accompanied corneal ulcerative processes.The local application of aprotinin (Trasylol, Bayer), an inhibitor of plasmin, and also of some other proteases, was found to be necessary for the healing of severe corneal injuries in which highly elevated plasmin activity in the tear fluid and inflammatory cellulization of the cornea occurred (severe damage). It was beneficial in cases in which medium plasmin activity occurred in the tear fluid and inflammatory changes in the cornea were not too extensive. If used very early after injury, aprotinin prevents the appearance of high plasmin activity in the tear fluid, reduces the invasion of inflammatory cells into the corneal stroma, and accelerates the healing. Even the corneal transparency is restored in many cases.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486–1496, 1975 and Wahle, J Biol Chem 266: 3131–3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources.     

A novel alkaline, thermostable, protease-free lipase from Pseudomonas sp.

An extracellular, alkali-tolerant, thermostable lipase was from a Pseudomonas sp. It had optimal activity at 65 °C and retained 75% of its activity at 65 °C for 90 min. The pH optimum was 9.6 and it retained more than 70% activity between pH 5 and 9 for 2 h. The culture broth was free of protease and, at 30 °C, the culture filtrate retained all the activity for at least 7 days, without any stabilizer. In shake flask culture, addition of groundnut oil (3 g l^–1) towards the end of growth phase increased the activity from 4 U ml^–1 to 8 ml^–1.     

Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine

We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.This work was supported in part by NIH grant GM-48595 and by a Grant-In-Aid from the American Heart Association, Missouri Affiliate (92-10517).     

Brain plasmin enhances APP alpha-cleavage and Abeta degradation and is reduced in Alzheimer's disease brains

The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the α-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain plasmin plays a preventive role in APP amyloidogenesis. Consistently, we show that brain tissue from Alzheimer’s disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer’s disease.     

Different secretory actions of pancreastatin in bovine and human parathyroid cells

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1–47) and bovine PST(32–47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1–100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca²⁺. Both peptides failed to alter the concentration of cytoplasmic Ca²⁺([Ca²⁺]_i) of bovine cells. Human PST(1–52) and PST(34–52) did not affect PTH release or [Ca²⁺]_i of parathyroid cells from patients with hyperparathyroidism, nor [Ca²⁺]_i of normal human parathyroid cells. Furthermore, bovine PST(1–47) and bovine PST(32–47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca²⁺]_i.     

Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney.

At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake.     

Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.