Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The heavy chain of myosin-ID isolated from Dictyostelium was identified as an in vitro substrate for members of the Ste20p family of serine/threonine protein kinases which are thought to regulate conserved mitogen-activated protein kinase pathways. Yeast Ste20p and Cla4p and mammalian p21-activated protein kinase (PAK) phosphorylated the heavy chain to 0.5-0.6 mol of Pi/mol and stimulated the actin-dependent Mg2+-ATPase activity to an extent equivalent to that of the Ste20p-like myosin-I heavy chain kinase isolated from Dictyostelium. PAK purified from rat brain required GTPgammaS-Cdc42 to express full activity, whereas recombinant mouse mPAK3 fused to glutathione S-transferase and purified from bacteria, and Ste20p and Cla4p purified from yeast extracts were fully active without GTPgammaS-Cdc42. These results suggest, together with the high degree of structural and functional conservation of Ste20p family members and myosin-I isoforms, that myosin-I activation by Ste20p family protein kinases may contribute to the regulation of morphogenetic processes in organisms ranging from yeast to mammalian cells.
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PMID:Activation of myosin-I by members of the Ste20p protein kinase family. 894 16

The transfer of phospholipids across membrane bilayers is protein-mediated, and most of the established transporters catalyze the energy-dependent efflux of phospholipids from cells. This work identifies and characterizes a lysophospholipid transporter gene (lplT, formally ygeD) in Escherichia coli that is an integral component in the 2-acylglycerophosphoethanolamine (2-acyl-GPE) metabolic cycle for membrane protein acylation. The lplT gene is adjacent to and in the same operon as the aas gene, which encodes the bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-acyl carrier protein synthetase. In some bacteria, acyltransferase/acyl-ACP synthetase (Aas) and LplT homologues are fused in a single polypeptide chain. 2-Acyl-GPE transport to the inside of the cell was assessed by measuring the Aas-dependent formation of phosphatidylethanolamine. The Aas-dependent incorporation of [3H]palmitate into phosphatidylethanolamine was significantly diminished in deltalplT mutants, and the LplT-Aas transport/acylation activity was independent of the proton motive force. The deltalplT mutants accumulated acyl-GPE in vivo and had a diminished capacity to transport exogenous 2-acylglycerophosphocholine into the cell. Spheroplasts prepared from wild-type E. coli transported and acylated fluorescent 2-acyl-GPE with an apparent K(d) of 7.5 microM, whereas this high-affinity process was absent in deltalplT mutants. Thus, LplT catalyzes the transbilayer movement of lysophospholipids and is the first example of a phospholipid flippase that belongs to the major facilitator superfamily.
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PMID:Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a transporter (LplT) belonging to the major facilitator superfamily. 1566 33

Biosynthesis of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3'wzx, wzy, and waaL truncations were fused to a phoA-lacZalpha dual reporter capable of displaying both alkaline phosphatase and beta-galactosidase activity. The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. Our data revealed unique TM domain properties in these proteins, particularly for Wzx, indicating the potential for a charged pore. Novel periplasmic and cytoplasmic loop domains were also uncovered, with the latter in Wzy and WaaL revealing tracts consistent with potential Walker A/B motifs.
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PMID:Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures. 2082 6

The lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF of Staphylococcus aureus encompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast, Clostridium perfringens encodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins of C. perfringens in an S. aureus mprF deletion mutant, we found that both lipids can be efficiently produced in S. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the corresponding C. perfringens enzyme did not affect susceptibility to CAMPs such as nisin and gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.
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PMID:Alanyl-phosphatidylglycerol and lysyl-phosphatidylglycerol are translocated by the same MprF flippases and have similar capacities to protect against the antibiotic daptomycin in Staphylococcus aureus. 2249 94

Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.
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PMID:Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells. 2515 35