Comparison of antigen immunoassay and reverse transcriptase assay for monitoring human immunodeficiency virus infection in an antiviral trial.

We compared the Abbott enzyme immunoassay for human immunodeficiency virus (HIV) antigen with the reverse transcriptase assay (RTA) as a means of monitoring HIV infection during an antiviral trial. The Abbott enzyme immunoassay detected HIV earlier than RTA whether or not the patients were antigenemic and appears to be superior to RTA for detecting HIV in cultures used for monitoring clinical trials.

Hydrodynamic studies on the association of cytochrome P-450.

Extended ultracentrifugation experiments have been performed on cytochrome P-450 LM2 from rabbit liver microsomes to analyse the behaviour of this membrane protein. The sedimentation coefficients and molecular weights vary between 11 S and 19 S and 350,000 and 700,000, respectively. No concentration dependence of these values could be observed between 0.3 microM 5.0 microM protein. The sedimentation coefficients and molecular weights appear to correlate with the heme content. Partial loss of heme leads to formation of aggregates of higher molecular weight. Analysis of the distribution of sedimentation coefficients clearly reveals the heterogeneity of the individual samples with values ranging from about 8 to 23 S. Thus, the smallest particle in the associate mixture of the enzyme consists of at most 3 monomers.

Determination of the partial specific volume of cytochrome P-450 as a model of a membrane protein.

Cytochrome P-450 has been used as a representative of membrane proteins in investigations for experimental determination of its partial specific volume. Diverse methods were employed: sucrose density gradient centrifugation, sedimentation velocity measurements in media of varying density, as well as sedimentation equilibrium studies in H2O and D2O. The results are compared with each other and with the theoretically calculated value. As a reason for discrepancies preferential solvation is discussed, and recommendations are given for the methodical approach to determine the partial specific volume and, hence, the molecular weight of a membrane protein.

Physicochemical studies of the 7 S complex of rat liver ribosomes and its components.

The 7 S complex of rat liver ribosomes contains 5 S RNA and one protein (L-5). The molecular weights of these two components are 40 600 and 37 200, respectively, as estimated by analytical ultracentrifugation. Th complex stability under different solvent conditions was analyzed from the point molecular weights using a sedimentation equilibrium technique and computer stimulation. High concentrations of KCl and urea induce partial dissociation, whereas MgCl stabilizes the complex.

Biochemical and ultrastructural analysis of SV40 chromatin.

SV40 chromatin can be isolated in two forms: At moderate ionic strength (mu = 0.1-0.3) it contains histone H1 in addition to the four nucleosomal histones and has a highly condensed appearance in the electron microscope, being composed of a few closely connected large spheres [190 A (160, 220) diameter]. At high ionic strength (mu = 0.6-0.8) or after prolonged exposure to very low ionic strengths (mu less than 0.02), the compact form unfolds and the chromatin shows a typical nucleosomal morphology. Native SV40 DNA-protein complexes contain a median number of 24 nucleosomes. The number of superhelical turns does not differ in DNA obtained from the compact and the unfolded forms of chromatin. DNA-relaxing enzyme is found associated with SV40 chromatin and is capable of acting both on extraneously added circular DNA and on its own DNA in the nucleoprotein complex. Purified DNA-relaxing enzyme forms transiently nicked DNA intermediates where the enzyme can be found covalently attached to the site of the nick in the DNA. Transcriptionally active SV40 complexes undergo the same ionic-strength-dependent structural transition as that of bulk SV40 chromatin and may therefore also have a compact configuration at physiological salt concentrations.

Stepwise relaxation of supercoiled SV40 DNA.

We have shown in this paper that agarose gel electrophoresis can be used to examine the tertiary structure of supercoiled SV40 DNA. Differences as small as one superhelical turn can be detected by this method. When used as an assay, it enabled us to purify from human tissue culture cells a protein which acts on supercoiled DNA, leading to a gradual removal of its superhelical turns. Using in turn the purified DNA relaxing protein to generate reaction intermediates of DNAs with different numbers of superhelical turns, we deduced that native SV40 DNA contains a minimum of 20 physical superhelical turns. The upper limit of this number is less accurately defined, but we think that it is not higher than 24. These numbers agree within very close limits with the numbers determined previously by various indirect physical methods.