Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subcellular localization of plasminogen activator (PA) in human neutrophils was studied. The cells were disrupted by nitrogen cavitation and fractionated on Percoll density gradients into three major components containing the plasma membranes, the specific granules, and the azurophilic granules. The biochemical markers we used to identify these organelles were alkaline phosphatase, vitamin B12-binding protein, and beta-glucuronidase, respectively. Using the radioactive fibrin plate method, PA activity and plasminogen-independent fibrinolytic activity were measured. In resting neutrophils, PA was associated mainly with the membranes of the specific granules. In five individual experiments the activity of this fraction varied from 79 to 100% of the total; the remaining activity was found to be associated with the plasma membrane, and no activity was present in the azurophilic granules. In neutrophils that were activated by exposure to PMA (20 ng/ml for 15 min at 37 degrees C), the total recoverable PA activity remained unchanged; however, the main peak of activity (85% of total) shifted from the specific granules to the plasma membranes. The magnitude of the reduction of the enzyme in the specific granules paralleled that of vitamin B12-binding protein. PMA-activated, intact neutrophils had approximately 12-fold more surface-bound PA activity than resting cells. Recovery of PA activity from neutrophils was critically dependent on pretreatment of the intact cells with DFP before cavitation; 100-fold more PA activity was detected in DFP-pretreated cells. At the same time, this pretreatment reduced the plasminogen-independent fibrinolytic activity by approximately sevenfold. We determined that PA present in the neutrophils is of the urokinase (UK) type and that the enzyme is produced and stored as a pro-UK, a form insensitive to DFP inhibition. The reduction in the level of proteases (measured as fibrinolytic activity) and the resistance of pro-UK to DFP are most likely the two major reasons for the greatly improved recovery of PA from the DFP-pretreated cells. These findings show that in resting neutrophils PA is stored in the specific granules, and that during activation, it translocates to the outer surface of the plasma membranes, thus equipping the cell with an ecto-proteolytic potential.
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PMID:Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis. 374

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.
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PMID:Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis. 1175 93