Distribution of urokinase-type plasminogen activator immunoreactivity in the mouse.

Immunocytochemistry, using rabbit antibodies to a urokinase-type 48-Kdalton Mr mouse plasminogen activator, showed that enzyme immunoreactivity is widely distributed in the normal mouse. Strong staining was obtained in widely disseminated connective tissue cells with a fibroblast-like morphology. Such cells occurred in high numbers in the lamina propria mucosae of the gastrointestinal tract, and in moderate numbers in the connective tissue septa of the pancreas. A few such cells were detected around the larynx, trachea, and bronchi. Immunoreactivity also occurred in epithelial cells of the proximal and distal kidney tubules, the ductus deferens, and in pulmonary pneumocytes. In addition, presumably extracellular staining was seen irregularly along the basement membrane and fibrillar structures in the lamina propria of the small and large intestines. Moreover, decidual cells of the mouse placenta stained strongly, and a moderate staining was observed in epithelial cells of involuting mammary glands, but not in those of noninvoluting glands. No immunoreactivity was observed in endothelial cells. Control experiments included absorption of the antibodies against highly-purified mouse plasminogen activator and the corresponding proenzyme, and the finding of a good correspondence between the number of immunoreactive cells and measurable enzymatic activity determined in adjacent tissue sections. Separation by SDS PAGE followed by immunoblotting revealed only one immunochemically stainable protein band with Mr approximately 48 Kdaltons in extracts from tissues showing immunoreactivity.

Immunocytochemical localization of urokinase-type plasminogen activator in Lewis lung carcinoma.

The invasively growing and metastasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.

Temperature-induced changes in fatty acid unsaturation of Tetrahymena membranes do not require induced fatty acid desaturase synthesis.

The usual rise in phospholipid-bound palmitoleic acid of Tetrahymena pyriformis cells slowly acclimating to low temperature exposure can be prevented by cycloheximide. This reduction in fatty acid desaturation is not caused by specific inhibition of a temperature-induced synthesis of a fatty acid desaturase but rather by a general effect equally conspicuous in isothermal cells. Cycloheximide-inhibited cells chilled and analyzed quickly, before long term ill effects of the drug are expressed, exhibit the rise in unsaturated fatty acids typical of temperature-acclimating cells.

Environmental effects on Tetrahymena membranes. Temperature-induced changes in membrane fatty acid unsaturation are indepedent of the molecular oxygen concentration.

The study was designed to show whether the increase in unsaturated fatty acids in Tetrahymena pyriformis membrane phospholipids with decreasing growth temperature is due to the sharp rise in the concentration of dissolved O2 found at lower temperatures. Increasing cell density in cultures growing at a constant temperature was found to have a more drastic effect upon the O2 level than shifting a culture from one temperature extreme to the other. However, the pattern of phospholipid fatty acid unsaturation did not vary over a 30-fold gradual decrease in O2 concentration measured in cultures during logarithmic growth at 39.5 degrees C, and unsaturation in 15 degrees C cells sampled over a 5-fold decrease in O2 concentration was also unchanged. Cells grown at 39.5 degrees C under a constant relatively high O2 concentration (5--6 mg O2/l) or low O2 concentration (0.3 mg O2/l) had nearly identical distributions of membrane fatty acids. Only under almost completely anaerobic conditions was it possible to measure via radioactive labeling experiments and inhibition of fatty acid desaturation. Thus the increased membrane fatty acid unsaturation induced by decreasing growth temperature is caused by some factor other than the rising levels of dissolved O2.

Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1).

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) >> Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.

The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells.

The cDNA coding for the 246-amino acid long N-terminal extracellular portion of the human (h) GH receptor, corresponding to the circulating GH-binding protein (hGHBP), was cloned by polymerase chain reaction from human IM-9 lymphocytes. The cDNA sequence was identical to that reported for human liver and placenta and demonstrated alternative splicing of exon 3. The protein with the exon 3-encoded domain was expressed and secreted in glycosylated form from baby hamster kidney (BHK) cells, purified to homogeneity, and sequenced; the amino acid sequence was identical to that predicted from liver cDNA. The cloned hGHBP competed in a dose-dependent fashion for binding of 125I-labeled 22-kilodalton (kDa) hGH, and at higher concentrations for binding of 125I-labeled 20-kDa hGH, to IM-9 lymphocytes. hGHBP decreased the association rate of [125I]hGH to the cells without decreasing the dissociation rate. hGHBP blocked the down-regulation of GH receptor in IM-9 cells by both 22- and 20-kDa hGH. hGHBP also blocked the binding of [125I]hGH to PRL receptors on Nb2 lymphoma cells and the effect of the hormone on thymidine incorporation. Binding of both 22- and 20-kDa hGH to the binding protein was demonstrated directly by immunoprecipitation with monoclonal antibody 263. The present work thus establishes the identity of the IM-9 human GHBP from those of liver and placenta, and demonstrates its ability to bind both 22- and 20-kDa hGH with good affinity and to block their biological actions mediated though both somatogenic and lactogenic receptors. The modulation of receptor down-regulation by the BP may be a relevant facet of its physiological role.

Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass.

Purified approximately 54 kDa plasminogen activator inhibitor from human fibrosarcoma cells was converted to an inactive form with slightly higher electrophoretic mobility by incubation with catalytic amounts of urokinase-type or tissue-type plasminogen activator. Serine proteinase inhibitors and a monoclonal antibody against urokinase-type plasminogen activator inhibited the conversion, indicating that it was caused by plasminogen activator-catalyzed proteolysis. These findings represent the first demonstration of a well-defined protein apart from plasminogen, constituting a substrate for plasminogen activators.

Proenzyme to urokinase-type plasminogen activator in the mouse in vivo.

We have investigated whether urokinase-type plasminogen activator (u-PA) is present in the mouse in vivo as the proenzyme or as the active enzyme. u-PA in extracts of various murine tissues was of a one-polypeptide chain form with an electrophoretic mobility indistinguishable from purified proenzyme (pro-u-PA), as demonstrated by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblotting. No 2-chain u-PA was detected in any of the extracts (detection limit 10% of that of one-chain u-PA). In bladder urine more than half of the u-PA was of the one-chain form. Together with previous immunocytochemical studies of the normal murine tissues and studies of the Lewis lung carcinoma, the present results indicate that in these tissues the one-chain proenzyme is the predominant form of u-PA in intracellular stores and for the first time demonstrates that at least in some cases the one-chain form constitutes a sizeable fraction of the u-AP in extracellular fluids in the intact organism.

Regeneration of cilia in starved Tetrahymena thermophila involves induced synthesis of ciliary proteins but not synthesis of membrane lipids.

The synthesis of ciliary-membrane phospholipids and ciliary proteins was studied after deciliation in starving Tetrahymena thermophila cells. Deciliated cells regenerated the new ciliary membrane without any induced phospholipid synthesis. The constant cell volume found during the regrowth of the cilia suggests that renewal of ciliary membranes takes place by insertion of intracellular membrane material into the cell surface. In contrast with the absence of induced phospholipid synthesis during ciliary regeneration, the synthesis of ciliary proteins was found to be induced. This enhanced synthetic activity was made possible by an increased rate of intracellular protein degradation in regenerating cells. It was found that the extent of the induced synthesis strongly depends upon the growth conditions of the cells before starvation. Furthermore, it was shown that the degree of induced protein synthesis is greater for higher-molecular-weight ciliary proteins than for lower-molecular-weight species.

The relationship between energy-dependent phagocytosis and the rate of oxygen consumption in Tetrahymena.

The induction of high rates of food vacuole formation in Tetrahymena pyriformis increased the rate of respiration in exponentially growing cells by 17% and in starving cells by 47.5%. The increased rate of oxygen uptake was caused by phagocytosis itself, as shown by comparing the rates of respiration of a Tetrahymena mutant exposed to particles at the permissive or restrictive temperatures for food vacuole formation. During cell division, heat-synchronized cells in rich, particle-supplemented medium showed a significant decrease in the rate of respiration. Furthermore, dimethyl sulphoxide, in concentrations sufficient to block food vacuole formation, suppressed the rate of respiration to a level similar to that of starved cells. Cytochalasin B, fowever, did not reduce the rate of oxygen uptake despite the inability of the cells to complete the formation of food vacuoles during treatment; a possible explanation for this finding is discussed. There was a strong correlation between formation of food vacuoles and a high metabolic rate in Tetrahymena.

Altered ligand specificity of proteolysed insulin-like growth factor binding protein-3.

IGF binding protein-3 (IGFBP-3) undergoes limited proteolysis in human pregnancy serum, altering its electrophoretic mobility and its binding of radioiodinated IGF tracers. IGF-I tracer, monoiodinated on Tyr31, discriminated less than other tracers between intact and proteolysed IGFBP-3. In competitive binding curves using this tracer, intact IGFBP-3 showed comparable reactivity towards IGF-I and analogs with substitutions at Tyr24, Tyr31 or Tyr60. In contrast, proteolysed IGFBP-3 reacted equally with IGF-I and [Ala31]IGF-I (approximately 10-fold lower potency than with intact IGFBP-3), but showed a marked selectivity against [Ser24]IGF-I and [Leu60]IGF-I (approximately 100-fold lower potency than with intact IGFBP-3). The affinity of IGF-I binding to proteolysed, but not intact, IGFBP-3 was increased by the addition of the acid-labile subunit of the IGFBP-3 complex. This study defines more fully the lesion in IGFBP-3 caused by serum proteolysis during pregnancy and demonstrates that tyrosine-substituted IGF-I derivatives are valuable tools in studying IGFBP-3 proteolysis.

Plasminogen activator released as inactive proenzyme from murine cells transformed by sarcoma virus.

We have previously reported the purification of a plasminogen-activating serine protease with an approximate Mr of 48 000 from sarcoma-virus-transformed murine cells. We now report that under serum-free conditions the enzyme is released from the cells in an inactive form. After affinity chromatography with 4-aminobenzamidine-cellulose, ion-exchange chromatography and gel filtration, the proenzyme could be obtained from culture fluid as a pure, homogeneous protein as evaluated by polyacrylamide gel electrophoresis with sodium dodecylsulphate. Proenzyme was quantitatively converted to active enzyme by incubation with catalytic amounts of plasmin. Analysis by polyacrylamide gel electrophoresis with sodium dodecylsulphate under reducing and non-reducing conditions showed that the inactive form consisted of a single polypeptide chain with an Mr of approximately 48 000, while the active form consisted of two chains with Mr values of approximately 18 000 and 29 000 , held together by one or more disulphide bridges. The active-site reagent diisopropylfluorophosphate in radiolabelled form was incorporated into the 29 000-Mr chain of the active enzyme, but not into the inactive form. These findings provide conclusive evidence for the existence of an inactive proenzyme to this plasminogen activator and thus demonstrate and additional step in a cascade-like reaction leading to extracellular proteolysis. Regulatory as well as methodological implications of this findings are discussed.

A simple, rapid immunometric assay for determination of functional and growth hormone-occupied growth hormone-binding protein in human serum.

We present a sensitive time-resolved fluorometric immunofunctional assay (TR-FIA) for direct quantitation of functional growth hormone-binding protein (GHBP), using an immunoassay kit for growth hormone (GH-DELFIA). In addition to the immobilized GH antibody, one monoclonal antibody against GHBP was used. This anti-GHBP was labelled with the chelate of europium. The assay was performed in one step. The detection limit for GHBP was 0.044 nmol L-1 (NBS + 3 SD). The calibration curve was linear in the interval 0.11-8.03 nmol L-1. Average intra-assay coefficient of variation (CV) was 3.44%. Average interassay CV at GHBP concentrations 0.563 nmol L-1 and 1.40 nmol L-1 were 12% and 6.3% respectively. Analytical recovery in serum ranged from 76% to 127% with a mean of 101 +/- 3.6%. Serum GHBP in 102 normal subjects ranged from 0.513 to 3.772 nmol L-1 and was positively related to body mass index (P < 0.001). In growth hormone-deficient sera GHBP was higher than in control subjects (1.751 +/- 0.179 nmol L-1 and 1.257 +/- 0.140 nmol L-1 respectively, P < 0.001). Acromegalic patients had lower levels of GHBP than controls (0.946 +/- 0.251 and 1.234 +/- 0.144 nmol L-1 respectively, P = 0.005). This assay also allowed detection of GH-complexed GHBP in serum. These results were in agreement with theoretical values calculated from the measured GH and the functional GHBP concentrations. Results were compared with data obtained by a recently reported, validated ligand immunofunctional assay (LIFA), which is fundamentally different. There was a significant linear relationship between the results from the two assays (r = 0.89, P = 0.001). The slope of the regression line was 0.65. In conclusion, this new convenient GHBP TR-FIA provides a sensitive and precise method for detecting total GHBP as well as complexed GHBP in human serum, and allows easy processing of large numbers of samples.

One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity.

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.

Molecular control of membrane properties during temperature acclimation. Fatty acid desaturase regulation of membrane fluidity in acclimating Tetrahymena cells.

This is a study of the molecular mechanisms employed by Tetrahymena pyriformis to change the lipid composition and thereby the fluidity of its various membranes during temperature acclimation. By quantitatively measuring the intramembrane particle aggregation using freeze-fracture electron microscopy, membrane physical properties in 39.5 degrees C grown cells shifted to 15 degrees C were found to be correlated with the degree of phospholipid fatty acid desaturation. Alteration of the phospholipid polar head group distribution from that of 39.5 degrees C-grown cells to the significantly different pattern of 15 degrees C grown cells appeared not to be of critical importance in the acclimation process. Changes in fatty acid desaturation during acclimation from high to low temperatures and vice versa were analyzed using normal cells and cells fed large amounts of polyunsaturated fatty acids. Fatty acid desaturase activity corresponded to the degree of membrane fluidity but not to the cell temperature. All evidence was compatible with the hypothesis that membrane fluidity is self-regulating, with the action of fatty acid desaturases being modulated by the physical state of their membrane environment.

Molecular control of membrane properties during temperature acclimation. Membrane fluidity regulation of fatty acid desaturase action?

Further studies on the molecular mechanisms of temperature acclimation have been carried out using the ciliate Tetrahymena pyriformis. The most prominent change in lipid metabolism during acclimation to high temperature--depression of fatty acid desaturase activity--could be simulated by supplementing the growth medium of isothermally-grown cells with polyunsaturated fatty acids. Such cells resisted the membrane-fluidizing effect of the incorporated exogenous acids by increased use of de novo synthesized saturated acids in their phospholipids. The data support the conclusions arising from earlier experiments with temperature-shifted cells (Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., and Thompson, G.A., Jr. (1976), Biochemistry 15), showing that, when membrane fluidity increased to a superoptimal level, the activity of membrane-associated fatty acid desaturases was decreased. Since the reaction is controlled by membrane fluidity, rather than temperature per se, we postulate that it is the general mechnaism employed by cells adjusting to any fluidity-modifying factor, such as cations, drugs, etc.

Plasminogen activator inhibitor from human fibrosarcoma cells binds urokinase-type plasminogen activator, but not its proenzyme.

An approximately 75% pure form of a human Mr approximately 54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose. The inhibitor inhibited human urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator, but not plasmin. Rabbit antibodies against this plasminogen activator inhibitor also reacted with a plasminogen activator inhibitor with identical electrophoretic mobility in extracts of human blood platelets, indicating that the HT-1080-inhibitor is of the same type as the inhibitor of blood platelets. As revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin-agarose zymography, incubation of HT-1080-inhibitor with the active form of human u-PA led to the formation of an equimolar sodium dodecyl sulfate-resistant complex between them; in contrast, no complex formation was observed between the inhibitor and the proenzyme form of human u-PA (pro-u-PA). Likewise, using a column of anti-inhibitor antibodies coupled to Sepharose for removal of excess inhibitor and activator-inhibitor complexes, the potential enzymatic activity of pro-u-PA was found to be unaffected by incubation with inhibitor under conditions in which more than 95% of the active u-PA had formed complex with inhibitor.

Enzyme-linked immunosorbent assay for human urokinase-type plasminogen activator and its proenzyme using a combination of monoclonal and polyclonal antibodies.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was greater than 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.