Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

Studies on the cell-cycle dependency of the actions of insulin-like growth factor-I in Balb/c 3T3 cells

The present study was conducted to determine the cell-cycle dependency of various actions of IGF-I in Balb/c 3T3 cells. When autophosphorylation of the IGF-I receptor was determined in [32P]-labelled cells, IGF-I increased radioactivity in a 100 K-Da phosphoprotein, presumably beta-subunit of the IGF-I receptor, both in quiescent and in primed competent cells. Likewise, IGF-I stimulated uptake of [3H]deoxyglucose independent of the cell cycle. In contrast, IGF-I increased calcium entry, radioactivity in [3H]diacylglycerol, and [3H]thymidine incorporation in primed competent cells while these reactions were not induced by IGF-I in quiescent cells. The latter three reactions were attenuated when cells were pretreated with pertussis toxin. These results indicate that some, but not all, of the actions of IGF-I are dependent on the cell cycle in Balb/c 3T3 cells. They also suggest that a pertussis-toxin-sensitive G protein may be involved in the cell-cycle-dependent actions of IGF-I.     

Synthesis of polypeptides by microwave heating I. Formation of polypeptides during repeated hydration-dehydration cycles and their characterization

Summary Amino acid amides effectively reacted to produce polypeptides in response to microwave heating during repeated hydration-dehydration cycles. The polypeptides, formed from a mixture of glycinamide, alaninamide, valinamide, and aspartic acid -amide, had molecular weights ranging from 1000 to 4000 daltons. Amino acids were incorporated into the polypeptides in proportion to the starting concentrations, with the exception of glycine whose incorporation was 1.5 times higher than that of the other amino acids. The polypeptides had some definite secondary structure, such as -helix and -sheet, in aqueous solution. This reaction provides not only a convenient method for abiotic peptide formation but also a convenient method for the chemical synthesis of peptides.     

Purification and characterization of a peptide C-terminal alpha-amidating enzyme from porcine atrium

In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. Here we report the purification and characterization of an alpha-amidating enzyme from porcine atrium, in which a high concentration of alpha-amidating activity was detected. The enzyme was purified to homogeneity from the membrane fraction of porcine atria by five steps of chromatography, including an affinity chromatography using a Sepharose column coupled with a substrate, Tyr-Phe-Gly. The purified enzyme was found to be composed of a single polypeptide chain with an apparent molecular weight of 92,000. This enzyme converted several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides.     

Enhancement of plasminogen activator activity in cultured endothelial cells by granulocyte colony-stimulating factor

A hitherto unknown function of granulocyte colony-stimulating factor (G-CSF) was found using cultured endothelial cells. G-CSF stimulated activity of plasminogen activator (PA) in both extracellular and intracellular milieus of endothelial cells obtained from bovine carotid and aortic artery. This effect was dependent on the concentration of G-CSF added to the culture medium and on the treatment time. The extracellular activity was enhanced approximately 5-fold at a concentration of 5,000 colony-forming unit (CFU)/ml (2.6 nM) and in about a 15-hr treatment period. Analyses by fibrin and reverse fibrin autography revealed that activity of PA was much more increased than that of PA inhibitor in endothelial cells treated with G-CSF.     

Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.     

Multi-recognition capability of E-selectin in a dynamic flow system, as evidenced by differential effects of sialidases and anti-carbohydrate antibodies on selectin-mediated cell adhesion at low vs. high wall shear stress: a preliminary note.

E-selectin has a "multi-recognition" capability in terms of epitope binding specificity, depending on adhesion conditions (static vs. low- or high-shear stress dynamic systems). Specifically, (i) adhesion based on expression of alpha 2-->3 sialylated Le(x) (SLe(x)) is prominent under static or low shear stress dynamic conditions; (ii) adhesion under high shear stress dynamic conditions does not depend on the known SLe(x) species, but rather on Lex with an adjacent unidentified sialosyl substitution, which shows different susceptibility to sialidases and antibodies compared to known SLe(x).     

Effects of activin A and somatostatin on intact FSH secretion and intracellular Ca2+ concentration in human FSH-secreting pituitary adenoma cells.

Activin A stimulated synthesis and secretion of intact FSH in dispersed human FSH-secreting adenoma cells. Significant stimulation was observed after 24 hr. Activin A caused an increase in Ca2+ concentration ([Ca2+]i). This response occurred soon after the activin A action. These effects were blocked in Ca(2+)-deficient medium and by nitrendipine (5 microM). Somatostatin inhibited the activin A-induced intact FSH secretion and the [Ca2+]i response. These findings indicated that Ca2+ influx through voltage-gated Ca2+ channel was involved in the activin A induced synthesis and secretion of intact FSH.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.