Interferon-induced proteins. Purification and characterization of a 15,000-dalton protein from human and bovine cells induced by interferon

Human interferons induce a protein of 15,000 daltons in human and bovine cells. This protein is located in the cytoplasm in a soluble form and is induced by concentrations of interferon which induce the antiviral state. Messenger RNA prepared from interferon-treated human and bovine cells contains a mRNA which yields on translation in vitro a protein similar in size to the 15-kDa protein induced by interferon in vivo. The human protein has been purified to homogeneity from interferon-treated human cells by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. A comparison of the peptides generated by V8 protease from the human and bovine 15-kDa proteins reveals that the two proteins are similar but not identical.     

Cleavage of Viral Precursor Proteins In Vivo and In Vitro

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.     

低温对植物叶片中超氧物歧化酶、过氧化氢酶和过氧化氢水平的影响

番茄和鸡蛋果叶片中可提取的SOD活性不受低温的影响。在电泳谱带上SOD主同工酶带被氰化物而不被低温抑制,次同工酶带在低温下不稳定,且活性很低,它的变化不影响总的SOD活性。一些冷敏感植物叶片中CAT活性被低温抑制,而H_2O_3水平在低温下稳定或有增加,这可能使毒性更强的羟基离子(OH·)易于形成。     

Cleavage of Poliovirus-Specific Polypeptide Aggregates

Zonal electrophoresis resolves two aggregates of poliovirus type 2 cytoplasmic polypeptides. The more negatively charged aggregate contains mainly noncapsid viral-specific polypeptides (NCVP) 2 and x, whereas the other consists of the capsid polypeptides (VP) 0, 1, 2, and 3 (VP0, VP1, VP2, VP3). After treatment with sodium deoxycholate (DOC), the aggregates sediment at 5 to 6S. Their electrophoretic mobilities are unaffected by DOC or RNase. The capsid polypeptide aggregate is similar in mobility to virions but can be converted to a faster electrophoretic form, resembling empty capsids, by heating. If infected HeLa cells are allowed to synthesize poliovirus polypeptides in the presence of iodoacetamide, no capsid polypeptides are produced, but rather NCVP1a (the precursor to capsid polypeptides) is accumulated, along with NCVP2 and NCVPx. When analyzed by electrophoresis and centrifugation, uncleaved NCVP1a migrates with the NCVP2-x aggregate. NCVP1a can be cleaved to capsid-like polypeptides in vitro by using extracts of infected cells, but not uninfected cells, indicating either a virus-specified protease or a cellular enzyme activated during infection. After cleavage of NCVP1a by infected cell extracts, the capsid polypeptides which are produced dissociate from the NCVP2-x complex.     

Zonal electrophoresis and isoelectric focusing of proteins and virus particles in density gradients of small volume

Proteins and virus particles were separated by zonal electrophoresis or isoelectric focusing in glass tubes of small volume. The tubes were covered at the bottom with dialysis membranes and sucrose gradients containing either buffer or ampholytes were generated directly into them. When ampholytes were employed, reproducible pH gradients were generated during electrophoresis. After the separations were finished, dense sucrose was pumped into the bottom of each tube and the gradient was fractionated from the top; the recovery of virus was nearly complete.