An isocitrate lyase of higher plants: analysis and comparison of some molecular properties

A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.     

Analysis of human metaphase chromosomes using antibodies to double-stranded and single-stranded DNA: staining patterns are related to DNA conformation

Fresh and 6-day-old fixed chromosome spreads, both untreated and treated with various banding techniques and nucleases, were stained using monoclonal antibodies to double-stranded and single-stranded DNA. DNA in fixed chromosome preparations became progressively denatured with ageing. The staining pattern of untreated chromosomes with anti-double-stranded DNA antibodies (which resembles G-banding) was determined by the conformation of the chromosomal DNA.     

Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

Time-resolved emission spectra of hemoglobin on the picosecond time scale

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.     

Analysis of electric and magnetic fields leaking from induction heaters

Results are presented of an investigation on electric and magnetic fields leaking from inductive (magnetic) heaters that are used for thermal processing of high-power electron tubes and lasers in an industrial plant. Measurements of electric and magnetic fields were done using both commercially available and laboratory-developed instrumentation. Isotropic H-field sensors were developed to allow quantitative evaluation of high-intensity magnetic fields. Ten induction heaters with nominal A.C. power ranging from 2.5 kW to 15 kW and operating at frequencies between 300 kHz and 790 kHz were surveyed. Electric field strengths up to 8 kV/m and magnetic field strengths up to 20 A/m were measured.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.     

Standardization of factor VIII: establishment and use of secondary standards

Two secondary standards for use in routine assays of Factor VIII in therapeutic concentrates and in patients, plasmas, respectively, have been established in a multicenter collaborative study. In order to assess the effect of the adoption of these preparations as common Secondary Standards a comparative assay has been performed: one sample of a Factor VIII concentrate of intermediate purity and one plasma sample have been tested in two laboratories for Factor VIII:C activity using as reference, among others, the common working standard. Analysis of the results shows that with the plasma sample the differences of the estimates obtained with any of the references in our two laboratories were not statistically significant (P greater than 0.3), while with the concentrate sample the differences were always statistically significant (P less than 0.005). The study shows that the adoption of common working standards (besides the uniformity in assay method, reagents and basic equipment) is not sufficient to eliminate interlaboratory variation in the measurement of Factor VIII:C.     

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

Specific antibodies in children excreting cytomegalovirus

Acute-and convalescent-phase sera from 22 children were examined by ELISA in comparison with a routine complement fixation (CF) test for detection of anti-CMV antibodies. All these subjects were excreting CMV from urine and/or saliva. The results showed that ELISA is more sensitive than CF test. Particularly ten children showed, by ELISA, anti-CMV antibody titers more agreeing with clinical-virological features. Generally, in other subjects the results of the two serological tests were similar. Three cases showed discordances both between the two methods and between serological data and clinical virological findings.