Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

Modelled female sale options demonstrate improved profitability in northern beef herds.

OBJECTIVE: To examine the impact of improving the average value of cows sold, the risk of decreasing the number weaned, and total sales on the profitability of northern Australian cattle breeding properties. DESIGN: Gather, model and interpret breeder herd performances and production parameters on properties from six beef-producing regions in northern Australia. PROCEDURE: Production parameters, prices, costs and herd structure were entered into a herd simulation model for six northern Australian breeding properties that spay females to enhance their marketing options. After the data were validated by management, alternative management strategies were modelled using current market prices and most likely herd outcomes. RESULTS: The model predicted a close relationship between the average sale value of cows, the total herd sales and the gross margin/adult equivalent. Keeping breeders out of the herd to fatten generally improves their sale value, and this can be cost-effective, despite the lower number of progeny produced and the subsequent reduction in total herd sales. Furthermore, if the price of culled cows exceeds the price of culled heifers, provided there are sufficient replacement pregnant heifers available to maintain the breeder herd nucleus, substantial gains in profitability can be obtained by decreasing the age at which cows are culled from the herd. CONCLUSION: Generalised recommendations on improving reproductive performance are not necessarily the most cost-effective strategy to improve breeder herd profitability. Judicious use of simulation models is essential to help develop the best turnoff strategies for females and to improve station profitability.

Fine root chemistry and decomposition in model communities of north-temperate tree species show little response to elevated atmospheric CO2 and varying soil resource availability.

Rising atmospheric [CO2] has the potential to alter soil carbon (C) cycling by increasing the content of recalcitrant constituents in plant litter, thereby decreasing rates of decomposition. Because fine root turnover constitutes a large fraction of annual NPP, changes in fine root decomposition are especially important. These responses will likely be affected by soil resource availability and the life history characteristics of the dominant tree species. We evaluated the effects of elevated atmospheric [CO2] and soil resource availability on the production and chemistry, mycorrhizal colonization, and decomposition of fine roots in an early- and late-successional tree species that are economically and ecologically important in north temperate forests. Open-top chambers were used to expose young trembling aspen (Populus tremuloides) and sugar maple (Acer saccharum) trees to ambient (36 Pa) and elevated (56 Pa) atmospheric CO2. Soil resource availability was composed of two treatments that bracketed the range found in the Upper Lake States, USA. After 2.5 years of growth, sugar maple had greater fine root standing crop due to relatively greater allocation to fine roots (30% of total root biomass) relative to aspen (7% total root biomass). Relative to the low soil resources treatment, aspen fine root biomass increased 76% with increased soil resource availability, but only under elevated [CO2]. Sugar maple fine root biomass increased 26% with increased soil resource availability (relative to the low soil resources treatment), and showed little response to elevated [CO2]. Concentrations of N and soluble phenolics, and C/N ratio in roots were similar for the two species, but aspen had slightly higher lignin and lower condensed tannins contents compared to sugar maple. As predicted by source-sink models of carbon allocation, pooled constituents (C/N ratio, soluble phenolics) increased in response to increased relative carbon availability (elevated [CO2]/low soil resource availability), however, biosynthetically distinct compounds (lignin, starch, condensed tannins) did not always respond as predicted. We found that mycorrhizal colonization of fine roots was not strongly affected by atmospheric [CO2] or soil resource availability, as indicated by root ergosterol contents. Overall, absolute changes in root chemical composition in response to increases in C and soil resource availability were small and had no effect on soil fungal biomass or specific rates of fine root decomposition. We conclude that root contributions to soil carbon cycling will mainly be influenced by fine root production and turnover responses to rising atmospheric [CO2], rather than changes in substrate chemistry.

Influence of adjunct cultures on volatile free fatty acids in reduced-fat Edam cheeses.

The effects of the adjunct cultures Lactococcus lactis ssp. diacetylactis, Brevibacterium linens BL2, Lactobacillus helveticus LH212, and Lactobacillus reuteri ATCC 23272 on volatile free fatty acid production in reduced-fat Edam cheese were studied. Lipase activity evaluation using p-nitrophenyl fatty acid ester substrates indicated that L. lactis ssp. diacetylactis showed the highest activity among the 4 adjunct cultures. Full-fat and 33% reduced-fat control cheeses (no adjunct) were made along with 5 treatments of reduced-fat cheeses, which included individual, and a mixture of the adjunct cultures. Volatile free fatty acids of cheeses were analyzed using static headspace analysis with 4-bromofluorobenzene as an internal standard. Changes in volatile free fatty acid concentrations were found in headspace gas of cheeses after 3-and 6-mo ripening. Acetic acid was the most abundant acid detected throughout ripening. Full-fat cheese had the highest relative amount of propionic acid among the cheeses. Certain adjunct cultures had a definite role in lipolysis at particular times. Reduced-fat cheese with L. lactis ssp. diacetylactis at 3-mo showed the highest levels of butyric, isovaleric, n-valeric, iso-caproic, and n-caproic acid. Reduced-fat cheese with Lactobacillus reuteri at 6 mo produced the highest relative concentration of isocaproic, n-caproic, and heptanoic, and the highest relative concentration of total acids.

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels.

AIMS/HYPOTHESIS: The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (KATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated. the specificity of repaglinide for three types of cloned (KATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of KATP channel, respectively. METHODS: The action of the drug was studied by whole-cell current recordings of KATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. RESULTS: The drug blocked all three types of KATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 micromol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2-8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. CONCLUSION/INTERPRETATION: Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the KATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo.

Human follistatin-related protein: a structural homologue of follistatin with nuclear localization.

Follistatin-related protein is a recently discovered glycoprotein that is highly homologous in both primary sequence and exon/intron domain structure to the activin-binding protein, follistatin. We explored their potential for functional redundancy by investigating the relative affinities and kinetics of their interactions with activin, bone morphogenic protein-6, and bone morphogenic protein-7 and by exploring their expression and distribution in human tissues and cells. Follistatin and follistatin-related protein mRNA were ubiquitous by Northern analyses, although their sites of peak distribution differed, with follistatin-related protein and follistatin predominating in the placenta and ovary, respectively. Follistatin-related protein, like follistatin, preferentially bound activin with high affinity and in an essentially irreversible fashion. Although follistatin-related protein, like follistatin, possesses a signal sequence and no known nuclear localization signals, its secretion was undetectable in most cell lines by RIA. Intriguingly, follistatin-related protein was identified as a nuclear protein in human granulosa cells and all human cell lines tested. Furthermore, Western analyses of CHO cells transfected with human follistatin-related protein revealed this protein to reside within the insoluble nuclear protein fraction. We conclude that despite its remarkably high level of similarity to follistatin with regard to structure and activin binding kinetics, follistatin-related protein is a nuclear as well as a secretory protein that may perform distinct intracellular actions.

Twisted gastrulation can function as a BMP antagonist.

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal-ventral development. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG), modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal-ventral patterning, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.

Peroxisome proliferator-activated receptor gamma target gene encoding a novel angiopoietin-related protein associated with adipose differentiation.

The nuclear receptor peroxisome proliferator-activated receptor gamma regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARgamma ligands, termed PGAR (for PPARgamma angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis.

The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein.

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.

Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Structure and functional expression of a human interleukin-8 receptor.

Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.

Selection for transformation and met protooncogene amplification in NIH 3T3 fibroblasts using tumor necrosis factor alpha.

Alterations in the structure and expression of protein tyrosine kinases are associated with cellular transformation and tumorigenicity. These enzymes may contribute to tumor progression by interfering with host mechanisms of antitumor surveillance. In the present work, we show that selection of NIH 3T3 fibroblasts for resistance to the cytotoxic effects of tumor necrosis factor alpha (TNF-alpha), a major mediator of macrophage-induced tumor cell cytotoxicity, leads to enrichment in the remaining cells for those with transformed morphology and is often associated with amplified copy number and expression of the met protooncogene. We further substantiate the relationship between met protooncogene amplification and resistance to TNF-alpha by showing that spontaneous (non-TNF-alpha-selected) NIH 3T3 cell transformants which have amplified met protooncogene copy number have increased resistance to the growth-inhibitory activity of this cytokine. These results provide evidence for one mechanism by which the activated macrophage may select for tumor cells within a developing focus with properties associated with tumor progression. In addition, our ability to select cells with such properties, as demonstrated using the met protooncogene as a model system, may also provide a unique means (i.e., selection with TNF-alpha) for identifying other gene products, including other tyrosine kinases, associated with aggressive tumor growth.

Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator.

The cDNA encoding a low Mr derivative (residues 144-411) of human single-chain urokinase-type plasminogen activator was cloned, the recombinant low Mr single-chain urokinase-type plasminogen activator (rscu-PA-32k) was expressed in Chinese hamster ovary cells, and the translation product was purified to homogeneity from conditioned cell culture medium. rscu-PA-32k is very similar to intact recombinant single-chain urokinase-type plasminogen activator in terms of its very low activity (120 IU/mg) on a chromogenic substrate for urokinase (pyroglutamylglycylarginine p-nitroanilide), its plasminogen-dependent fibrinolytic activity on fibrin plates (specific activity = 170,000 IU/mg), its plasminogen activating potential, and the lack of specific binding to fibrin. In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared to low molecular weight two-chain urokinase (50% lysis at 2.1 and 1.6 mg/kg infused over 4 h). Thrombolysis was associated with much less extensive systemic fibrinogen breakdown with rscu-PA-32k than with two-chain urokinase (residual fibrinogen at 50% lysis of 71 and 10%, respectively). It is concluded that the functional properties of rscu-PA-32k, expressed with a high efficiency, are similar to those of its previously characterized natural counterpart.

Enzymatic properties of single-chain and two-chain forms of a Lys158----Glu158 mutant of urokinase-type plasminogen activator.

Recombinant single-chain urokinase-type plasminogen activator (rscu-PA), in which the plasmin-sensitive peptide bond Lys158-Ile159 is destroyed by site-specific mutagenesis of Lys158 to Glu (rscu-PA-Glu158), is quantitatively converted to two-chain urokinase-type plasminogen activator (rtcu-PA-Glu158) by treatment with endoproteinase Glu-C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu-PA-Glu158 for the activation of plasminogen is 20 times lower (0.0001 microM-1 s-1) than that of rscu-PA (0.002 microM-1 s-1). In contrast, rtcu-PA-Glu158 has very similar properties to rtcu-PA obtained by digestion of rscu-PA with plasmin, including binding to benzamidine-Sepharose, catalytic efficiency for the activation of plasminogen (0.035 microM-1 s-1 versus 0.046 microM-1 s-1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single-chain urokinase-type plasminogen activator but not of the two-chain form.

Alpha 2-antiplasmin Enschede: alanine insertion and abolition of plasmin inhibitory activity.

An abnormal alpha 2-antiplasmin that is associated with a serious bleeding tendency has been found in a Dutch family and is referred to as alpha 2-antiplasmin Enschede. This abnormal alpha 2-antiplasmin is converted from an inhibitor of plasmin to a substrate. The molecular defect of alpha 2-antiplasmin Enschede, as revealed by sequencing of cloned genomic DNA fragments, consists of an alanine insertion near the active site region of the molecule. Substitution of this fragment into complementary DNA for a wild-type alpha 2-antiplasmin yields a translation product with physical and functional properties typical of the abnormal alpha 2-antiplasmin Enschede. The naturally occurring mutant may serve as a model for investigating the structures that determine the properties of an inhibitor versus those of a substrate in serine protease inhibitors.

Characterization of recombinant human alpha 2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site.

Human alpha 2-antiplasmin (alpha 2AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r alpha 2AP) is immunologically identical with natural alpha 2AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k1) for the interaction of alpha 2AP and r alpha 2AP with plasmin are both (1-2) X 10(7) M-1 s-1. In order to examine the effects of alterations within the reactive site of alpha 2AP, deletions of the P1 residue Arg-364 (r alpha 2AP-delta Arg364) or the P'1 residue Met-365 (r alpha 2AP-delta Met365) were introduced by in vitro site-directed mutagenesis. r alpha 2AP-delta Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that alpha 2AP has no absolute requirement for Met in the P'1 position. Unexpectedly, no increase in antithrombin activity was observed. r alpha 2AP-delta Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor (k1 = 1.5 X 10(5) M-1 s-1).

Amino-acid sequence of human alpha 2-antiplasmin.

The amino-acid sequence of human alpha 2-antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature alpha 2-antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659-1664], 444 residues were identified by amino-acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369). alpha 2-Antiplasmin contains two disulfide bridges (Cys64-Cys104 and Cys31-Cys113) and four glucosamine-based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. alpha 2-Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.

Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator.

A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411. The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA. The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000. On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA). The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase. Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA. The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma. Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA. CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1). It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.

Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158.

The cDNA encoding full-length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced, and the recombinant scu-PA (rscu-PA) was expressed in Chinese hamster ovary cells. Two mutants, constructed by in vitro site-specific mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158) or to Glu158 (rscu-PA-Glu158), were also expressed in Chinese hamster ovary cells. Wild type and mutant rscu-PAs were purified to homogeneity by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration. The specific activity of the mutant scu-PAs on fibrin plates is very low (less than 1,000 IU/mg) compared to that of the wild type rscu-PA (44,000 IU/mg). The mutants, in contrast to the wild type rscu-PA, are not converted to amidolytically active two chain u-PA (tcu-PA) by plasmin and do not cause lysis of a 125I-fibrin-labeled plasma clot immersed in citrated plasma. However, in a purified system, both rscu-PA-Gly158 and rscu-PA-Glu158 activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (Km = 60-80 microM) but with a higher turnover rate constant (k2 = 0.01 s-1) as compared to the wild type rscu-PA (Km = 1.0 microM, k2 = 0.002 s-1). We conclude that conversion of scu-PA to tcu-PA is not a prerequisite for the activation of plasminogen. Substitution of Lys158 by Gly158 or Glu158 does, however, markedly decrease the stability of the Michaelis complex.