Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell proliferation, tissue plasminogen activator (t-PA) production and metabolic changes of Hamster Kidney cells (HaK) grown on microcarriers in an automatic Dynamic Cell Culture System (DCCS) were determined on the first International Microgravity Mission (IML-1) Spacelab (22-30 January 1992). The DCCS was designed for two cell culture chambers (volume: 200 microliters each), one operating as a hatch system, the other as a perfusion system. Medium exchange was achieved with an osmotic pump (flow rate 1 microliter h-1). Two major items were investigated: the biological performance of the DCCS in space and the effect of microgravity on HaK cells. The results obtained demonstrated that (1) the DCCS can be used for biological experiments on long term Spacelab missions. In fact, higher cell densities and higher concentration of glucose but lower concentration of lactate in the perfusion chambers than in the batch chambers were measured. The concentration of t-PA, glutamine and ammonia was similar in all chambers. (2) Microgravity had no effect on cell growth and metabolism of HaK cells.
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PMID:Cultivation of hamster kidney cells in a dynamic cell culture system in space (Spacelab IML-1 Mission). 1154 89

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.
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PMID:Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. 1173 37

Thrombin-activatable fibrinolysis inhibitor (TAFI) circulates as an inactive proenzyme of a carboxypeptidase B-like enzyme (TAFIa). It functions by removing C-terminal lysine residues from partially degraded fibrin that are important in tissue-type plasminogen activator mediated plasmin formation. TAFI was classified as a metallocarboxypeptidase, which contains a Zn(2+), since its amino acid sequence shows approximately 40% identity with pancreatic carboxypeptidases, the Zn(2+) pocket is conserved, and the Zn(2+) chelator o-phenanthroline inhibited TAFIa activity. In this study we showed that TAFI contained Zn(2+) in a 1:1 molar ratio. o-Phenanthroline inhibited TAFIa activity and increased the susceptibility of TAFI to trypsin digestion. TAFIa is spontaneously inactivated (TAFIai) by a temperature-dependent intrinsic mechanism. The lysine analogue epsilon-ACA, which stabilizes TAFIa, delayed the o-phenanthroline mediated inhibition of TAFIa. We investigated if inactivation of TAFIa involves the release of Zn(2+). However, the zinc ion was still incorporated in TAFIai, indicating that inactivation is not caused by Zn(2+) release. After TAFIa was converted to TAFIai, it was more susceptible to proteolytic degradation by thrombin, which cleaved TAFIai at Arg(302). Proteolysis may make the process of inactivation by a conformational change irreversible. Although epsilon-ACA stabilizes TAFIa, it was unable to reverse inactivation of TAFIa or R302Q-rTAFIa, in which Arg(302) was changed into a glutamine residue and could therefore not be inactivated by proteolysis, suggesting that conversion to TAFIai is irreversible.
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PMID:Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor. 1180 20

Factor XIIIa (FXIIIa) catalyzes the covalent crosslinking of fibrin polymers and incorporation of proteins into the fibrin network and thus confers on the thrombus additional structural stability and relative resistance to plasmin-mediated degradation. Moreover, FXIIIa is involved in other physiological and pathophysiological processes such as wound healing and arteriosclerosis. Selective FXIIIa inhibitors may be a valuable tool for evaluation of the various functions of FXIIIa and their pharmacological control. This paper presents an overview of the inhibitors of FXIIIa. Analogues of natural FXIIIa substrates - including glutamine containing peptides and low molecular weight substituted alkylamines - are incorporated into the fibrin network and thus prevent crosslinking of fibrin. Naturally occurring, direct inhibitors of FXIIIa have been isolated from a leech species and microorganisms. With effective concentrations in the nanomolar range the peptide tridegin is the most potent FXIIIa inhibitor up to now. The majority of the synthetic, low molecular Weight inhibitors bind covalently to Cys314 at the active site of FXIIIa. Besides the relatively nonspecific thiol reagents, azol derivatives, azolium salts and related substances are described as specific inhibitors of FXIIIa. They inhibit the activity of FXIIIa at nanomolar concentrations. Animal experiments have demonstrated improved thrombolysis by a plasminogen activator in combination with a FXIIIa inhibitor.
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PMID:[Inhibitors of factor XIIIa]. 1219 84

The human group IIA secreted PLA(2) is a 14 kDa calcium-dependent extracellular enzyme that has been characterized as an acute phase protein with important antimicrobial activity and has been implicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interface plays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis, one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutants H48Q, H48N, and H48A had been prepared for the secreted PLA(2)s from bovine pancreas and bee venom and retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity. Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme that retained significant (2-4%) catalytic activity that was contrary to expectations in view of the accepted catalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions including assays in the presence of Co(2+) and Ni(2+) in place of Ca(2+). The crystallization of the H48Q mutant, yielding diffraction data to a resolution of 1.5 A, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins, and the catalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation of the calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than the anticipated full proton transfer to the histidine will best explain the ability of an active site glutamine to allow significant catalytic activity.
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PMID:The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism. 1250 Nov 75

Single-nucleotide polymorphisms (SNPs) are differences in the nucleotide sequence of a specific gene from different individuals. The frequency at which SNPs occur varies among individuals, is gene dependent, and may be influenced by the aging process or by mechanisms that result in cell transformation. Urokinase-plasminogen activator (uPA) is a serine protease that is important in embryonic development, aging, and the onset of pathogenic conditions. The frequency of SNP and the stability of the SNPs in the uPA gene have not been defined with regard to processes that are associated with cellular aging or transformation. In this study, the complete nucleotide sequence has been determined for the gene encoding uPA from 26 human diploid kidney cell lines. The frequency of SNP occurrence within the uPA gene and whether this frequency changed during cellular aging, or after cell transformation, were determined. The results demonstrated three donor-dependent SNPs. One SNP was located at base pair 422, which is in the region of the gene responsible for encoding the high-molecular weight domain of uPA (HMW-uPA). The other SNPs were located at base pairs 691 and 822, both of which are in the region of the gene responsible for encoding the low-molecular weight domain of uPA (LMW-uPA). Single-nucleotide polymorphisms were not detected in the portion of the gene responsible for encoding the uPA secretion signal. Leucine or proline would be encoded at amino acid 141 of HMW-uPA as the result of an SNP at base pair 422. The SNP detected at base pair 691 would encode for lysine or glutamine at amino acid 231 of LMW-uPA. The SNP detected at base pair 822 would not change the encoded asparagine located at position 274 of the protein. The SNPs identified in this study were donor dependent and were not altered during cellular aging, or by changes in karyology due to spontaneous transformation of the cell line. These results demonstrate that the integrity of the uPA gene is stable and not subject to alterations that accompany cell aging or transformation.
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PMID:Single-nucleotide polymorphism of the urokinase-plasminogen activator gene during aging and transformation of human diploid kidney cell cultures. 1468 74

A strategy for fed-batch cultivation of t-PA producing recombinant CHO cells is presented, based on the substitution of glucose and glutamine for slowly metabolized nutrients and in a rational design of the medium. Media for the batch and fed stages were based on the cell specific amino acid requirements, which allowed a more accurate determination of the initiation of the fed stage and the frequency of nutrient addition from then on. Salt concentration was also reduced in both media to avoid an increase in osmolality. As a consequence of this rational design, most amino acid did not accumulate significantly during the fed stage, as usually occurs when their supply is not based on cell requirements; also, lower amounts of by-products were obtained when osmolality level was kept low, that altogether increased viability, longevity and t-PA production when compared with a reference batch culture. Alternating glucose and galactose during the fed stage, allowed lactate detoxification of the cells through their own metabolism. This allowed an increase in cell growth and cell viability with respect to a fed-batch culture in which only glucose was used in the fed stage.
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PMID:Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. 1512 36

Fibrinogen is a large, complex, fibrous glycoprotein with three pairs of polypeptide chains linked together by 29 disulfide bonds. It is 45 nm in length, with globular domains at each end and in the middle connected by alpha-helical coiled-coil rods. Both strongly and weakly bound calcium ions are important for maintenance of fibrinogen's structure and functions. The fibrinopeptides, which are in the central region, are cleaved by thrombin to convert soluble fibrinogen to insoluble fibrin polymer, via intermolecular interactions of the "knobs" exposed by fibrinopeptide removal with "holes" always exposed at the ends of the molecules. Fibrin monomers polymerize via these specific and tightly controlled binding interactions to make half-staggered oligomers that lengthen into protofibrils. The protofibrils aggregate laterally to make fibers, which then branch to yield a three-dimensional network-the fibrin clot-essential for hemostasis. X-ray crystallographic structures of portions of fibrinogen have provided some details on how these interactions occur. Finally, the transglutaminase, Factor XIIIa, covalently binds specific glutamine residues in one fibrin molecule to lysine residues in another via isopeptide bonds, stabilizing the clot against mechanical, chemical, and proteolytic insults. The gene regulation of fibrinogen synthesis and its assembly into multichain complexes proceed via a series of well-defined steps. Alternate splicing of two of the chains yields common variant molecular isoforms. The mechanical properties of clots, which can be quite variable, are essential to fibrin's functions in hemostasis and wound healing. The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active enzyme plasmin, results in digestion of fibrin at specific lysine residues. Fibrin(ogen) also specifically binds a variety of other proteins, including fibronectin, albumin, thrombospondin, von Willebrand factor, fibulin, fibroblast growth factor-2, vascular endothelial growth factor, and interleukin-1. Studies of naturally occurring dysfibrinogenemias and variant molecules have increased our understanding of fibrinogen's functions. Fibrinogen binds to activated alphaIIbbeta3 integrin on the platelet surface, forming bridges responsible for platelet aggregation in hemostasis, and also has important adhesive and inflammatory functions through specific interactions with other cells. Fibrinogen-like domains originated early in evolution, and it is likely that their specific and tightly controlled intermolecular interactions are involved in other aspects of cellular function and developmental biology.
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PMID:Fibrinogen and fibrin. 1583 18

Ammonium is a toxic and inhibitory byproduct of mammalian cell metabolism. At the end of a typical recombinant protein production campaign, the ammonium concentration can be as high as 10 mM, mainly due to glutamine metabolism. Intracellular pH (pH(i)) levels are sensitive to ammonium, which negatively impacts both cell growth and recombinant protein productivity. Ammonium also negatively affects the recombinant protein glycosylation profile, thus altering quality. Many strategies have been adopted to reduce ammonium accumulation, with limited results. This study investigated the addition of amino acids to the growth media for Chinese hamster ovary (CHO) cell cultures as a means of mitigating the negative effects of ammonium. Threonine, proline, and glycine additions improved CHO cell growth and recombinant protein levels. Further, the threonine, proline, and glycine additions positively impacted important metabolic parameters, including glucose consumption, lactate production, glutamine utilization, and final ammonium levels. Additionally, threonine, proline, and glycine increased the level of alpha(2,3)-linked sialic acid, galactose-beta(1,4)-N-acetylglucosamine, and alpha(2,6)-linked sialic acid residues on the recombinant tissue plasminogen activator (t-PA). Thus, threonine, proline, and glycine can be used to mitigate some of the toxic effects of ammonium on cell growth, recombinant protein productivity, and protein quality.
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PMID:Effects of amino acid additions on ammonium stressed CHO cells. 1586 58

We report a novel fibrinogen variant (fibrinogen Seoul II), which has a heterozygous point mutation from CAA to CCA leading to AalphaGln328Pro. The mutation site is among several glutamine residues that serve as alpha-chain cross-linking acceptor sites. Fibrinogen Seoul II was found in a 51-year-old male patient and his family in Seoul, Korea. The patient was diagnosed with myocardial infarction at age 43. Eight years later he was admitted to the emergency room due to recurrence of the disease, where he expired under treatment with tissue plasminogen activator (t-PA). Fibrin polymerization curves, made using purified fibrinogen from the patient's relatives, showed a decreased final turbidity, suggesting Seoul II fibrin clots are composed of thinner fibers. This supposition was verified using scanning electron microscopy. Alpha-polymer formation by the mutant fibrinogen upon thrombin treatment in the presence of factor XIII and calcium was distinctly impaired. This result confirms that the residue Aalpha328 plays a pivotal role in alpha-chain cross-linking.
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PMID:A novel fibrinogen variant (fibrinogen Seoul II; AalphaGln328Pro) characterized by impaired fibrin alpha-chain cross-linking. 1673 2


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