Urokinase-type plasminogen activator in colorectal carcinomas and adenomatous polyps: quantitative expression of active and proenzyme.

Total urokinase-type plasminogen activator (u-PA) content (proenzyme plus active enzyme) was significantly higher in 20 colorectal carcinomas and in 27 adenomatous polyps than in metaplastic polyps and autologous normal mucosa. u-PA content was also markedly increased in adenomatous polyps and autologous colonic mucosa removed from familial polyposis coli patients. Using a new monoclonal antibody technique to distinguish the proenzyme of u-PA from the active enzyme, we found that 70% of the u-PA in polyp and cancer tissue was present in the proenzyme form compared to 47% in normal colonic mucosa. For colon cancers, there was a significant correlation between their stage of invasiveness and the levels of proenzyme. No correlation was observed between the u-PA content of adenomatous polyps and their size or degree of dysplasia. Study of the u-PA content of the colonic mucosa may offer a useful biochemical correlate of epithelial cell transformation in the colon.

Monoclonal antibody NMS-1 increases N-formyl chemotactic peptide-mediated oxidative burst generation in human neutrophils.

Murine monoclonal antibody (mAb) NMS-1 was generated which binds to the surface of living human neutrophils. The antigens on neutrophil plasma membranes recognized by mAb NMS-1 were solubilized in Nonidet P-40 and immunopurified on matrix-bound mAb NMS-1. mAb NMS-1 binds to four periodate-sensitive structures of 70,000, 95,000, 140,000, and 170,000 Da on the plasma membrane surface of human neutrophils as was shown by Western blot analysis. Binding of mAb NMS-1 to human neutrophils induced a rapid transient rise in cytosolic free calcium (Quin 2 fluorescence) but no detectable generation of reactive oxygen metabolites. The oxidative burst of N-formyl peptide-treated neutrophils, however, increased in the presence of mAb NMS-1. The kinetics of N-formyl peptide (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tryrosyl-lysine; FNLPNTL)-mediated hydrogen peroxide formation (p-hydroxy phenyl acetate oxidation) in the presence of mAb NMS-1 was analyzed quantitatively. 1) When neutrophils were incubated with mAb NMS-1 before FNLPNTL addition, an increase in rate, magnitude, and duration of hydrogen peroxide formation was observed compared with controls which received no mAb NMS-1 treatment. After termination of the initial linear phase of response, a second transient linear phase of hydrogen peroxide formation was induced. This second phase of activation was not observed in neutrophils which received no mAb NMS-1 treatment. The onset of the response and latency before attainment of the initial linear rate of hydrogen peroxide formation was not changed by mAb NMS-1 pretreatment. 2) When neutrophils were stimulated with FNLPNTL, the addition of mAb NMS-1--after termination of the FNLPNTL-induced response--without delay induced a second transient burst of hydrogen peroxide formation. Persistent activation of hydrogen peroxide formation by mAb NMS-1 in FNLPNTL-stimulated neutrophils was not observed.

Monoclonal antibodies inhibitory to human plasmin. Definitive demonstration of a role for plasmin in activating the proenzyme of urokinase-type plasminogen activator.

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high-Mr urokinase to its low-Mr form. None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti-plg 1 inhibited (a), (b) and (c), while anti-plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti-plg 3 and anti-plg 4 inhibited only activity (c). Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase-type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.

Minactivin expression in human monocyte and macrophage populations.

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.

Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution.

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.

The uncA gene codes for the alpha-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains.

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.

Properties of membranes from mutant strains of Escherichia coli in which the beta-subunit of the adenosine triphosphatase is abnormal.

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered beta-subunit of the magnesium ion-stimulated adenosine triphosphatase.

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.

Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli.

The effects of two protease inhibitors on the solubilization of the membrane-bound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, epsilon-amino-n-caproic acid, did not effect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solublized by the low-ionic-strength treatment.

The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria.

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.