A strong fibrinolytic enzyme was readily obtained in saline extracts of the earthworm, Lumbricus rubellus. It hydrolyzed not only plasminogen-rich fibrin plates, but also plasminogen-free fibrin plates. The average fibrinolytic activity was about 100 CU (plasmin units) or 250 IU (urokinase units)/g wet weight. The molecular weight and isoelectric point were about 20,000 and 3.4, respectively. The enzyme was heat-stable and displayed a very broad optimal pH range. DFP and SBTI strongly inhibited the enzyme, but the anti-plasmin agent, t-AMCHA, exerted little effect under the same conditions. Purification of the enzyme was performed and three partially purified fractions were obtained. These three fractions were further subdivided. The first fraction (F-I) was divided into three fractions (F-I-0, F-I-1, and F-I-2), which exhibited similar biochemical characteristics. The second fraction (F-II) could not be subdivided. The third fraction (F-III) was divided into two fractions (F-III-1 and F-III-2). Based on results for their enzymatic activities against various substrates, the fraction I enzymes are thought to represent a chymotrypsin-like enzyme and the fraction III enzymes to represent a trypsin-like enzyme. The fraction II enzyme appears to be neither a trypsin- or chymotrypsin-like enzyme nor an elastase. The amino acid compositions of the six enzymes were estimated. Compared with other serine enzymes, these enzymes contained very abundant asparagine or aspartic acid, and there was very little proline or lysine. From the above data, these enzymes are regarded as novel fibrinolytic enzymes, and we name them collectively as Lumbrokinase from the generic name of the earthworm.
We performed mass spectrometric imaging (MSI) to localize ginsenosides (Rb(1), Rb(2) or Rc, and Rf) in cross-sections of the Panax ginseng root at a resolution of 100 microm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Tandem mass spectrometry (MS/MS) of alkali metal-adducted ginsenoside ions revealed structural information of the corresponding saccharides and aglycone. MALDI-MSI confirmed that ginsenosides were located more in the cortex and the periderm than that in the medulla of a lateral root. In addition, it revealed that localization of ginsenosides in a root tip (diameter, 2.7 mm) is higher than that in the center of the root (diameter, 7.3 mm). A quantitative difference was detected between localizations of protopanaxadiol-type ginsenoside (Rb(1), Rb(2), or Rc) and protopanaxatriol-type ginsenoside (Rf) in the root. This imaging approach is a promising technique for rapid evaluation and identification of medicinal saponins in plant tissues.
The molecular structure of grayanotoxin XIX, C20H30O3, has been determined by means of X-ray crystal analysis. The crystals are triclinic, with two molecules in a unit cell with dimensions of a=10.406, b=10.648, c=8.003 Å, α=90.00, β=94.25, and γ=102.76°; the space group is P1. 2671 unique intensity data were collected on a four-circle diffractometer with Ni-filtered Cu Kα radiation. The structure was elucidated by the Monte Carlo direct method, using the 50 strongest reflections as the starting set. The block-diagonal least-squares refinement reduced the R value to 0.038. The structure obtained corresponds to 14-deoxygrayanotoxin VII. The two independent molecules exist in different conformations: one has a half-chair A-ring, and the other, an envelope A-ring. In both molecules, the B-ring takes a conformation intermediate between the chair and twist-chair forms, and the C-ring adopts a boat conformation. The crystal consists of infinite hydrogen-bonded molecular chains along the c axis.
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