Abstract: Catalytic antibody components, methods for producing catalytic antibody components, methods for using catalytic antibody components, in particular, single chain and smaller components are disclosed. Catalytic antibody components able to promote the cleavage or formation of an amide, peptide, ester or glycosidic bond, and which are prepared from monoclonal catalytic antibodies, catalytic autoantibodies or by site-directed mutagenesis are disclosed. Methods of using catalytic antibody components alone or in combination with other antibody components or other biological moieties are disclosed.

Abstract: Methods are disclosed for producing hybrid phospholipid-binding proteins from eukaryotic cells. DNA constructs comprising a transcriptional promoter, at least one signal sequence and a hybrid phospholipid-binding protein coding sequence comprising at least one lipocortin lipid-binding domain joined to a gla-domainless, vitamin K-dependent protein and a transcriptional terminator are also disclosed.

Abstract: A novel 170 kD membrane protease is isolated from malignant human melanoma cell line LOX and RPMI7951. The protease is useful in a method of diagnosing cellular transformation.

Abstract: A 46,000 Dalton thermostable, very acidic protease, which has been named thermopsin, was purified to homogeneity from the culture medium of Sulfolobus acidocaldarius by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, MonoQ (FPLC), and gel filtration (HPLC). The enzyme is a single polypeptide chain having proteolytic activity over pH range 0 to 11 at temperatures between 0.degree. C. and 100.degree. C., with maximal activity at approximately pH 2 and 90.degree. C. Antibodies directed against thermopsin have been prepared. Through studies using various aspartic protease inhibitors, thiol and metalloprotease inhibitors, and serine protease inhibitors, it was determined that, although similar to some aspartic proteases, the active site of thermopsin is clearly not identical to that of other aspartic proteases.

Abstract: An enzyme material isolated from a culture of Micrococcus sedentarius which comprises one or more proteases having an ability to degrade protein, including human callus. In one embodiment the enzyme material is water soluble, non-dialyzable through a membrane having a molecular weight cut-off of 10 kDa, has an isoelectric point of 4.6, a molecular weight of 30.3 kDa, an optimum pH for protease activity at about 8.2, and functions at an optimum temperature of about 40.degree. C. In another embodiment, the enzyme material is water-soluble, non-dialyzable through a membrane having a molecular weight cut off of 10 kDa, has an isoelectric point of 2.7, a molecular weight of 50 kDa, an optimum pH for protease activity at about pH 10.2, and functions at an optimum temperature of about 46.degree. C.

Abstract: Emulsifying agents are prepared by sequentially treating a biological material with a protease and with a lipase. The enzymatically treated biological material may be pasteurized during or following the enzymatic treatment.

Abstract: Novel chemically modified detergent enzymes are provided, wherein one or more methionines have been mutated into cysteines, said cysteines subsequently being chemically modified in order to confer the enzyme improved stability towards oxidative agents. A novel process for stabilizing detergent enzymes against oxidation is also provided. Furthermore, there are provided detergent compositions comprising these novel oxidation stable detergent enzymes.

Abstract: A hybrid plasminogen activator-like polypeptide comprising a polypeptide region responsible for an affinity to fibrin derived from a tissue plasminogen activator polypeptide and a polypeptide region responsible for an enzyme activity derived from a prourokinase polypeptide; a DNA segment coding for the hybrid polypeptide; plasmid containing the DNA segment; a microorganism transformed with the DNA; and a process for production of the hybrid polypeptide comprising culturing the microorganism and recovering the hybrid polypeptide from the cultured cells.

Abstract: A process for the purification of plasminogen activator inhibitor 2 (PAI-2) including the steps of preincubating a PAI-2-containing solution with a compound which cleaves disulfide linkages, mixing the solution with a water-soluble acridine or quinoline base to precipitate impurities in the solution, and separating the precipitated impurities to obtain PAI-2 in purified form.

Abstract: Fatty acid esters of methyl glycosides are prepared by reacting a fatty acid or ester with a methyl glycoside in the presence of an enzyme catalyst, in particular a lipase. The resulting fatty acid esters are preferably monoesters.The methyl glycoside fatty acid esters may be used as surface-active agents in cleaning compositions or personal care products.

Abstract: Antibacterially active compositions are prepared from a proteolytic enzymatic digestate of glycopeptides obtained from a protein isolate enriched with soya glycoprotein 7S or with bean glycoprotein II. The glycopeptide digestate is treated with endo-.beta.-N-acetylglucosaminidase H to obtain oligosaccharide compositions which then also may be treated with exo-.alpha.-mannosidase to obtain further oligosaccharide compositions.

Abstract: The invention concerns a peptidylhydroxyglycine N-C lyase (PHL) catalyzing the reactionR-GlyOH.fwdarw.R-NH.sub.2wherein R represents a peptide residue, and GlyOH represents an .alpha.-hydroxyglycine residue linked to the C-terminus of said peptide R by an amide bond, a recombinant DNA molecule coding for a PHL, a method for the preparation of such a recombinant DNA molecule, processes for the preparation of PHL from a natural source or by means of the said recombinant DNA molecule, and the use of PHL for the preparation of an amidated peptide R-NH.sub.2.

Abstract: A process for making L-aminoacylase includes a cultivation of microorganism selected from a specy of Alcaligenes, especially the Alcaligenes denitrificans DA 181, and a separation of a produced L-aminoacylase from the bacterial cells for obtaining the L-aminoacylase which may be further purified for the production of L-amino acid. The acylase made by such a process may have an increased stability, beneficial for its commercial and medical values.

Abstract: An exocellular protease from Thermomonospora fusca YX and a process for producing the protease which has the following physicochemical properties:(1) Molecular mass:The protease has a molecular mass of from about 10,000 to 14,000 Daltons as measured by SDS-polyacrylamide gel electrophoresis;(2) Influence of inhibitors:The protease activity is inhibited by serine protease inhibitors;(3) Substrate specificity:A non-specific protease which can hydrolyze food proteins and bovine serum albumin at a rate of 50-100 nmoles peptide bonds/.mu.g enzyme/minute at 55.degree., pH 8.5 in 0.5 M Tris buffer without showing any substrate and/or product inhibition;(4) Reactivity:A broad spectrum serine type protease having activity at least 5 times greater than trypsin or chymotrypsin towards food grade proteins and bovine serum albumin;(5) Optimum activity temperature and temperature range:The optimum activity temperature is 80.degree. C. at pH of 8.0 in 0.05 M Tris buffer at an ionic strength of 0.

Abstract: Compounds of the formula (R-COO).sub.n X-OR.sup.1, wherein R.sup.1 is optionally substituted alkyl, phenyl, or alkyl phenyl, n is 1, 2 or 3, X is a carbohydrate moiety, and R is optionally substituted alkyl, have superior effects as additives in detergents. These compounds can be prepared by esterification of glycosides using specific enzymes.

Abstract: Novel carbonyl hydrolase mutants derived from the DNA sequences of naturally-occurring or recombinant non-human carbonyl hydrolases are disclosed. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to generate the substitution of one or more amino acid residues in the amino acid sequence of a precursor carbonyl hydrolase. Such mutant carbonyl hydrolases have properties which are different from those of the precursor hydrolase and are especially useful in detergent formulations. The substituted amino acid residues correspond to position +123 and/or +274 in Bacillus amyloliquefaciens subtilisin.

Abstract: The blood-clotting protein, factor IX, is synthesized in the bod in liver cells, where it undergoes three distinct types of post-translational modification before it is secreted into the bloodstream as a 415 amino acid long protein. It is therefore a difficult protein to produce by recombinant DNA technology in a highly biologically active form. Nevertheless, such a result has been achieved by the present invention in which typically factor IX cDNA in a plasmid is linearized and inserted into an expression vector having a promoter sequence of SV40 early gene, an SV40 polyadenylation sequence, the TK/NEO selectable marker and an ampicillin resistance gene. Mammalian cells such as from a dog kidney or rat liver are transfected by the calcium phosphate precipitation method.

Abstract: A method for making a stable aqueous liquid formulation containing at least one enzyme having proteolytic activity is disclosed. The method involves precipitating the enzyme from an aqueous medium with a salt to form a dispersion of the enzyme. The dispersion has a density of 1.22 g/cm.sup.3 to 1.23 g/cm.sup.3 due to the use of the salt. This process yields a sedimentation-resistant stable enzyme dispersion.

Abstract: A chorionic peptidase-1 (C-ase-1) which is isolThe U.S. government may have rights concerning the present invention because relevant developmental work was supported by NIH grant No. HD 14842.

Abstract: A KEX2 endoprotease produced by a recombinant DNA technique, a KEX2 endoprotease shortened at the C-terminal of a native enzyme and still containing a C-terminal hydrophobic region, a soluble KEX2 endoprotease without a C-terminal hydrophobic region; DNA's coding for the above-mentioned enzymes; expression plasmids containing the DNA; hosts transformed with the plasmid; a process for production of the above-mentioned enzymes using the transformed host; and a process for production of biologically active polypeptide using the above-mentioned enzyme.

Abstract: An immunogenic composition for use as a vaccine against malaria. It comprises an enzymatically active protease with a molecular weight of 76.000 daltons, characterized by its capacity to react with protective antibodies originating from monkeys resistant to human malaria parasites, particularly to Plasmodium falciparum.

Abstract: Tissue plasminogen activator (tPA) species having a specific molecular weight can be isolated in a purified form from a crude tPA preparation containing various tPA species having different molecular weights by bringing the crude tPA preparation into contact with hydroxyapatite and then separately eluting the adsorbed tPA species with eluents having different pHs and/or salt concentrations.

Abstract: A novel and unique plasminogen activator inhibitor fragment is obtained from human umbilical vein endothelial cells which has the following characteristics:A. it is derived from a native t-PA inhibitor that binds to and inhibits the activity of t-PA,B. it is dissociated from a complex formed between said native t-PA inhibitor and t-PA, said complex existing in two distinct interconvertible conformations with molecular weight of about 88 KDa and 105 KDa, respectively, and being partially reversible in the presence of fibrin,C. it has a molecular weight of about 40 KDa when dissociated from the complex, andD. it has a novel partial N-terminal amino acid sequence when dissociated from the complex.

Abstract: Plasminogen activators (PA) are obtained from cultured normal human colon cells which are adaptable to large scale production. A purified tissue PA (t-PA) is obtained from CCD-18Co normal human colon fibroblast cells which shows chemical differences from Bowes melanoma t-PA.

Abstract: A method of quantitatively analyzing an analyte contained in a whole blood sample, wherein a dry multi-layered analysis element is used. The method provides particular merits when the used multi-layered analysis element has no light-shielding layer which is interposed, in the conventional analysis element, between a coloring reagent layer and a blood cell separating layer, so that red coloring matters of blood cells are detected from the support side during the step of measuring the optical density of the reflected light.

Abstract: A composition for thrombolytic therapy includes a tissue-type plasminogen activator (t-PA) and a low affinity heparin fraction. The composition is administered intravenously to allow the t-PA to dissolve blood clots while the low affinity heparin fraction prevents reocculsion without the harmful side effects observed for unfractionated heparin, such as, stimulation of and interference with t-PA activity in the circulatory system, as well as, interference with fibrinolytic activity which can cause hemorrhaging in the mammalian circulatory system.

Abstract: An analog-ligand having a conformation that substantially corresponds to the conformation of a hydrolytic transition state of an amide or ester reactant ligand is used to produce receptor molecules of predetermined specificity. The receptor molecules include an antibody combining site that binds to the analog-ligand and also to a reactant ligand and thereby stabilizes the tetrahedral carbon atom of the amide or ester hydrolysis transition state of that reactant ligand to catalytically hydrolyze the reactant ligand at a predetermined site.

Abstract: A gene encoding an enzyme aqualysin I is disclosed. The gene of the present invention comprises a region encoding a signal peptide; a pro-region located downstream the signal peptide-encoding region; a region encoding aqualysin I, which is located downstream the pro-region; and a tail region of aqualysin I, which is located downstream the aqualysin I-encoding region. This invention also provides a recombinant vector comprising the gene, Escherichia coli transformed with the recombinant vector and a process of producing aqualysin I utilizing the Escherichia coli.

Abstract: A biologically degradable film is prepared consisting of a synthetic polymer and a biologically degradable polymer. The biologically degradable polymer is divided into small particles by means of enzymes produced by microbes in the form of spores, which enzymes split and release small molecules from the surface of the biopolymer particles. After achieving desired particle size, an emulsion is formed with vegetable oil and particles coated with enzyme protein become coated with vegetable oil, which at the same time interrupts the degradation of the biopolymer particles by the enzyme. The coated particles with the oil is separated from the suspension to remove small molecules after which the particles are redried and then pulverized. The final film is prepared in a film extruder in which the biopolymer is mixed with the synthetic polymer and possibly with other additives that are generally used in forming polymer films.

Abstract: The present invention provides a one-step process for the detection of the presence of an allergy and for the specific detection of the allergen responsible for the allergy, in which the leukocytes of a sample to be investigated are incubated with an allergen or with another stimulation factor in an aqueous medium together with a chromogenic protease substrate and calcium ions, the liberated protease is reacted with the chromogen and the resulting chromophor is determined. The protease activity is measured kinetically after an incubation period by the increase of the chromophor concentration. The present invention also provides a reagent and a device for carrying out this process, as well as a process for the determination of antiallergic and anti-inflammatory substances. The device consists of a microtiter plate with a plurality of different reagents arranged in rows.

Abstract: A novel tissue plasminogen activator having the following characteristics: molecualr weight of 65,000-72,000 Daltons as measured by SDS-PAGE electrophoresis using at 7.5% agarose gel; plasminogen activator specific activity of about 10.4.times.10.sup.4 IU/mg, wherein specific activity is defined as the ratio of fibrinolytic activity of purified t-PA measured on fibrin-agarose plates to milligrams of protein; about 83.1% absorption of t-PA by a fibrin-Sepharose column when applied; binds to a Concanavalin A column when applied; the fibrinolytic activity is substantially undiminished by heating at 60.degree. C. for 60 minutes or 95.degree. C. for 5 minutes relative to unheated t-PA; unreactive with polyclonal antisera raised against urokinase; the fibrinolytic activity is substantially stable at pH 5-10; exhibits fibrinolytic activity at pH 7.5-9.0 and temperature 39.degree.-41.degree. C.; a Km value of about 1.16.times.10.sup.-3 mol/liter and a V.sub.max of about 11.7.times.10.sup.

Abstract: Crude tPA containing, as an impurity, a protein which is reactive with an anti-human tPA antibody and has a molecular weight of 110,000.+-.20,000 daltons is purified by bringing the crude tPA into close contact with an affinity reagent containing an immobilized Kunitz inhibitor which is produced in seeds of Erythrina latissima and other Erythrina plants and acts as an inhibitor on trypsin, plasmin and tPA but does not act on urokinase, so that tPA is collected selectively.

Abstract: A cysteine proteinase characterized in that it is immunologically distinct, has a molecular mass of about 25,000 and has a distinct net charge is disclosed. "Ananain" and "comosain" are such enzymes, which may be distinguished by their specificities for certain synthetic substrates.A process for the production thereof is also disclosed which comprises separation from pineapple plant material and purification. Such enzymes may be used in the debridement of eschar tissues.

Abstract: A modified t-PA having improved in vivo half-life is disclosed comprising in sequence a sequence of two K2 kringle regions and a serine protease region SP.

Abstract: A process for producing fructose-1,6-diphosphate comprising the steps of: (a) enzymatically converting adenosine 5'-diphosphate to adenosine 5'-triphosphate using an acetate kinase-containing microorganism or an extract of the microorganism and phosphate donor; and (b) enzymatically converting a substrate capable of being converted to glucose or fructose to fructose-1,6-diphosphate using the adenosine 5'-triphosphate resulting from step (a) and the acetate kinase-containing microorganism or the extract of the microorganism.

Abstract: A method of increasing the specific activity of tissue plasminogen activator is disclosed which comprises increasing the proportion of neutral oligosaccharides in the tissue plasminogen activator glycoprotein.

Abstract: Improved secretion of heterologous proteins by hosts such as yeast by using promoters of at most intermediate strength with heterologous DNA secretion signal sequences is disclosed. A promoter of at most intermediate strength, such as the actin (ACT) or iso-1-cytochrome c (CYC1) promoter in S. cerevisiae is operatively linked to a DNA signal sequence, such as the Mf.alpha.1 signal sequence. A DNA sequence for a selected protein, such as somatomedin C (SMC), tissue plasminogen activator (TPA) or tumor necrosis factor (TNF), may be operatively linked to the DNA signal sequence.

Abstract: Eucaryotic cells cotransformed with product and selection genes yield considerably greater quantities of product after a novel subcloning strategy is employed: Transformants are identified for product yield, cultured under selection pressure and the progeny screened for product yield. Novel transformation vectors contain directly ligated selection and product genes and/or eucaryotic promoters.

Abstract: The present invention provides a process for the activation of gene-technologically produced, biologically active proteins expressed in prokaryotes after cell digestion by solubilization under denaturing conditions and reducing conditions and subsequent reactivation under oxidizing and renaturing conditions, wherein working is carried out at a protein concentration of 1 to 1000 .mu.g./ml. and, between the solubilization and the reactivation, a dialysis is carried out against a buffer with a pH value of from 1 to 4 containing 4 to 8 mole/liter guanidine hydrochloride or 6 to 10 mole/liter urea.

Abstract: A screening procedure is provided which utilizes a milk clotting assay for selecting supersecreting yeast cells for obtaining high yields of desired polypeptide products.Supersecreting yeast cells are provided as filed with American Type Culture Collection.Final polypeptide products are obtained from mutant yeast strains which have been screened as to secreting properties with supersecreters then cultured to obtain high yields.

Abstract: There is disclosed a protease having the following enzymatic properties in view of:(a) function and substrate specificity,(b) optimum pH,(f) activation, and(g) inhibition;(a) the protease can hydrolyze, in particular, a peptide bond on the C-terminal side of Y of a peptide X--Y--, in which X is Arg, Lys or Pro optionally having a peptide bond on the N-terminal side, Y is Arg and -- indicates a peptide bond;(b) the protease has an optimum pH of about 7.0 in Tris hydrochloride buffer;(f) the protease is activated with calcium chloride or a surfactant; and(g) the protease is inhibited with p-amidinophenylmethanesulfonyl fluoride, p-chloromercuribenzoic acid, a metal chelater, tetraacetic acid or a heavy metal.

Abstract: The thrombolytic effect of known plasminogen activators such as tissue plasminogen activator (t-PA), prourokinase and modifications of t-PA and prourokinase is enhanced dramatically and unexpectedly when combined with streptokinase. The body is treated with a combination streptokinase and one or more plasminogen activators of the t-PA or prourokinase type to achieve an enhanced effect which has advantage in thrombolytic therapy including the treatment of myocardial infarction.

Abstract: Crystalline subtilisin is produced by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution (at least about 40 g/1). This process does not produce amorphous subtilisin even at high salt concentrations in the solution. Optionally, subtilisin seed crystals also may be added to the concentrate to speed up the crystallization process.

Abstract: Disclosed herein are improved processes for preparing variant human t-PA proteins exhibiting improved pharmacokinetic properties relative to natural t-PA. One such illustrated variant, devoid of amino acids corresponding to amino acids 1 through 44 of natural t-PA, is shown to exhibit a plasma half-life of greater than about 15 times the plasma half-life to natural t-PA, as well as a clearance rate of less than about 1/10 the clearance rate of natural t-PA. Also disclosed are improved processes for treating vascular disease employing pharmaceutical compositions which incorporate therapeutically effective amounts of such t-PA variants with pharmaceutically acceptable diluents or excipients.

Abstract: A modified tissue plasminogen activator having an improved in vivo half-life characterized in that the normal protein moiety of 527 amino acids is mutated at the site Cys73.fwdarw.Arg and at the site Lys277.fwdarw.Asp.

Abstract: The use of T7 bacteriophage to produce DNA length standards by enzymatically joining terminally repetitious, blunt-ended DNA has now been demonstrated. It is now possible to precisely control the formation of concatemeric DNAs thereby generating custom-made size-ranges of length standards. Furthermore, the standards thus produced are stable over time providing a highly reproducible and convenient product for the molecular biologist.

Abstract: A pharmaceutical composition is prepared wherein a biologically active conjugated plasminogen activator is selectively conjugated to at least one heparin fragment having a terminal 2,5-anhydro-D-mannose residue which has an aldehyde not involved in intramolecular hemiacetal formation. The resulting conjugate has a prolonged half-life as compared to native tissue plasminogen activator and is able to deliver heparin to the site of clot dissolution to prevent reocclusion.

Abstract: An enzyme system for hydrolysis of protein material from plant and animal sources is obtained by extracting germinated sorghum seeds. The enzyme system contains a proteolytic component active on intact proteins and large peptides at an optimum pH of 3.2 to 4.2 and a peptidase component active on small peptides at an optimum pH of 4.5 to 6.5. A protein hydrolyzate having an average molecular weight within a selected range can be obtained by varying the pH so as to depress activity of the proteolytic component or the peptidase component. The protein hydrolysates which are obtained are characterized by the absence of bitter taste. Furthermore, by suitably adjusting the enzyme concentration in the reaction medium, the temperature and the hydrolysis time, protein hydrolyzates with an average molecular weight lower than 700 and a low percentage of free amino acids can be obtained.

Abstract: An affinity gel for the isolation of serine proteases from a biological sample. The affinity gel is modified for specificity for serine proteases by coupling an organophosphorous compound to the gel. The reaction is carried out in two steps. The hydrophilic gel is reacted with a phosphoryl trifluoride and then the coupled gel is further reacted with an alcohol to form the organophosphorous compound.

Abstract: A method of separating activated human protein C, which comprises bringing a mixture containing activated human protein C having a gamma-carboxyglutamic acid (Gla) domain in the presence of a calcium ion into contact with a fixed antibody comprising an insoluble carrier and fixed thereto an antibody to a complex of human protein C and a calcium ion bound to the Gla domain whereby the activated human protein C is captured by the fixed antibody in the form in which the calcium ion is bound to the Gla domain.