A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate

This work reports a precipitation and differential protein solubility approach using saturated ammonium sulfate solutions as a depletion and fractionation approach for shotgun proteomic analysis of plasma samples.

Improved radioimmunoassay of melatonin in serum.

Melatonin was extracted from serum by using Baker reversed-phase C-18 columns. More than 99% of the applied melatonin was retained by the columns, and more than 97% was eluted from the columns in 300 microL of methanol. We then determined melatonin in the serum extract by a modification of a standard radioimmunoassay, using filtration instead of centrifugation to collect the [3H]melatonin-antibody complex precipitated by saturated ammonium sulfate. These modifications allow more rapid, accurate, and reproducible determination of melatonin than do previously published procedures.

MECHANISMS OF PLATELET AGGREGATION BY ACIDIC MUCOPOLYSACCHARIDE EXTRACTED FROM STICHOPUS JAPONICUS SELENKA IN HUMANS AND RABBITS

It has been reported that acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus selenka) (SJAMP) induced the aggregation of human and animal platelets by an unknown mechanism, using platelet-rich plasma (prp) and washed human and rabbit platelets we studied the effects of storage, platelet inhibitors, and various plasmas and their fractions on SJAMP-induced platelet aggregation. we found that the lowest concentrations of SJAMP required for aggregation of human and rabbit platelets were 0.4 and 2 ug/ml respectively. The reactivity of human platelets to SJAMP decreased with time after drawing of blood; rabbit platelets did not show this phenomenon. Platelet inhibitors such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose, and EDTA inhibited by 50 to 100% the aggregation of human platelets induced by SJAMP; but these inhibitors had no effect on SJAMP-induced aggregation of rabbit platelets. Washed human and rabbit platelets were not aggregated by SJAMP. The aggregation of washed human platelets by SJAMP was restored completely by human or rabbit plasma, by human fibrinogen, or by 0 to 30% saturated ammonium sulfate fraction but not by serum. The aggregation of rabbit platelets by SJAMP could only be restored by rabbit plasma or serum, or by 50 to 60% saturated ammonium sulfate fraction. The data indicate that the mechanisms of aggregation of human and rabbit platelets by SJAMP are different. THe SJAMP-induced human platelet aggregation is dependent upon metabolism, release of ADP and the cyclooxygenase pathway requiring fibrinogen and Ca++. The aggregation of rabbit platelets induced by SJAMP is independent of metabolism, release of ADP and cyclooxygenase pathway, and does not require fibrinogen and Ca++, but needs certain protein(s) in the 50 to 60% saturated ammonium sulfate fraction of rabbit plasma.

Crambe Thioglucoside Glucohydrolase (EC 3.2.3.1): Separation of a Protein Required for Epithiobutane Formation

Sonicated extract of crambe seed meal prepared in the presence of ferrous ion and dithiothreitol enzymatically converts epi-progoitrin to glucose, HSO4−, and a mixture of 1-cyano-2-hydroxy-3,4-epithiobutanes (50–70%) and 1-cyano-2-hydroxy-3-butene (30–50%). A fraction of the extract precipitating between 60 and 70% saturated ammonium sulfate contains thioglucosidase that converts epi-progoitrin essentially to 1-cyano-2-hydroxy-3-butene. Chromatography (on cross-linked dextran) of a 40–60% ammonium sulfate fraction leads to separation of a proteinaceous material (s20 = 2.6 S) that does not hydrolyze epi-progoitrin but, in the presence of thioglucosidase, promotes the formation of 1-cyano-2-hydroxy-3,4-epithiobutanes in amounts proportional to those from crude seed meal extract.

Selektive Anreicherung von Staphylokokken-Panton-Valentine-Leukozidin mit Aluminiumoxid-Cholesterin / Selective Enrichment of Staphylococcus “Panton-Valentine”-Leukocidine with Aluminium Oxide Cholesterol

A selective concentration of “Panton-Valentine”-leukozidine (PVL) from the culture-supernatant Staphylococcus aureus was achieved through adsorption of most of the a-hemolysin, coagulase, eggyolk-opacity factor and fibrinolysin to aluminiumoxide cholesterol. PVL was further concentrated through dialysis against a saturated ammonium sulfate solution and subsequent pressure-filtration. Through pressure-filtration relatively low-molecular staphylococcal substances, such as a part of a nuclease, could be separated from PVL.

Electron Microscopic Observations of L-Lactate Dehydrogenase Crystals

Dogfish muscle lactate dehydrogenase (LDH) has been shown by M. G. Rossmann and his associates at Purdue University to crystallize in space group 1422 with the tetramolecular tetragonal unit cell dimensions of a = b = 103.5, c = 155.1 Å. They have found the roughly cubic molecule to contain four approximately rectangular-prism-shaped subunits arranged in 222 symmetry. The molecular weight (140,000) and unit cell size suggest that these crystals might profitably be studied with the electron microscope for comparison with the x-ray information. Suitably sized crystals grown by Rossmann's group in about 40% saturated ammonium sulfate were either cross-linked with gluteraldehyde and embedded in Maraglas for sectioning or washed in 50% saturated ammonium sulfate preparatory to making the shadowed carbon replicas.