Biomedical subjects
Publications and source records attributed to R B Luftig.
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One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.
The effects of microtubule-depolymerizing agents on virion production in MJD-54 cells chronically infected with Moloney murine leukaemia virus were examined. By measuring the amount of reverse transcriptase activity remaining in particles recovered from culture fluids, we found that incubation with 1 microM- or 10 microM-colchicine, vinblastine or nocodazole resulted in 30 to 40% decreases in virus production. The decrease in virus production did not seem to be due to general damage to the cells since cellular RNA and protein synthesis were only slightly, if at all, inhibited by the drug treatment (less than 10%). Furthermore, virus proteins accumulated inside drug-treated cells, viz, [35S]methionine-labelled Pr65gag showed a 1.5-fold increase over a 3 h continuous label interval. Consistent with this accumulation of virus protein, electron microscope studies showed that inside drug-treated cells ther was a 2- to 2.5-fold accumulation of virions within cytoplasmic vesicles. All of these results support the idea that cytoplasmic microtubules play a role in the production of murine leukaemia virus.
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The effect of vinblastine on the distribution of murine leukemia virus-derived membrane-associated antigens was examined by using the indirect immunofluorescence of 3.7% formaldehyde-fixed MJD-54 (Moloney murine leukemia virus-infected) cells. On fixed, non-drug-treated cells, p30 antigen was distributed homogeneously and diffusely over the cell membrane. When cells were incubated with 10 microM vinblastine for 1 hr before fixation, the distribution of p30 antigen was greatly changed, fluorescence now being collected into poles (cap-like formation). In contrast to this distribution pattern for p30 antigen, gp70 antigen was distributed in a micropunctate pattern on the cell surface, with or without vinblastine pretreatment. These observations indicate that the distribution patterns of p30 and gp70 membrane antigens are completely different and that they are differently controlled by cytoplasmic microtubules. In addition, because the p30 membrane antigen visualized in these studies most likely represents viral Pr65gag precursor molecules which are localized directly under and associated with the plasma membrane, these results suggest that, under special conditions of fixation, it is possible to obtain a cap-like phenomenon for cytoplasmic (internal) membrane-oriented proteins.
Although the Pr65gag precursor polyproteins of Moloney murine leukaemia virus (M-MuLV) and of Rauscher murine leukaemia virus (R-MuLV) have the same apparent mol. wt. by SDS--polyacrylamide gel electrophoresis (SDS--PAGE), their initial approximately 40 000 dalton intermediate cleavage products differ in mol. wt., i.e. the M-MuLV product (Pr41.5gag) is 1500 daltons larger than the R-MuLV product (Pr40gag). We took advantage of this difference to show that in vitro cleavage of R-MuLV Pr65gag by the M-MuLV proteolytic activity gives rise to R-MuLV Pr40gag and not M-MuLV Pr41.5gag. This result suggests that the specificity for cleavage of the MuLV Pr65gag is built into the substrate.
Cleavage of murine leukemia virus Pr65gag is associated with the activity of a labile proteolytic factor found in virions. We have shown that the presence of 80 to 100 mM NaCl inhibits this cleavage activity in vitro by over 90%. Further, the addition of 80 to 100 mM excess NaCl in vivo to chronically infected cultures of MJD-54 mouse fibroblasts also caused inhibition of Pr65gag cleavage. Specifically, the excess salt added to cells: (i) caused a greater than 90% decrease in virus production; (ii) increased the Pr65gag/p30 ratio in virions produced by more than threefold; and (iii) in pulse-chase experiments, showed a 10-fold decrease in the amount of Pr65gag cleaved after 3 h. In contrast, during this chase interval there was only a slight diminution, i.e., about two fold, in the cleavage of env precursor polyprotein Pr80env, suggesting that cleavages of Pr65gag and Pr80env are differently controlled. Additionally, electron microscopic examination of the excess salt-treated cells showed a twofold increase in the number of associated immature particles, consistent with the observed higher than average Pr65gag/p30 ratio. The inhibitory effects were also found if excess KCl or MgCl2 was used instead of NaCl, suggesting that they are caused by the hypertonic state of the medium and are not dependent on the ionic species used.
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Microtubules from suspension cultures of HeLa cells have been purified by carrying them through four complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C. These microtubules show, in addition to the major alpha- and beta-tubulin components, major proteins with molecular weights of 201 000-206 000 (comprising 4.5% of the total protein), proteins with molecular weights of 97 000, 100 000, 104 000, and 114 000 (together comprising approximately 2% of the total protein), and minor components with molecular weights of 68 000 and 151 000. HeLa microtubules have also been reconstituted from purified HeLa tubulin and proteins from HeLa microtubules separated from tubulin by DEAE-cellulose column chromatography. Experiments on the fractionation and reconstitution of both two- and four-cycle microtubules suggest that the 201 000-206 000-dalton proteins are incorporated into microtubules and promote tubulin polymerization. Microtubules formed by fractionationand reconstitution of two-cycle microtubules also contain several other proteins with molecular weights of 132 000, 146 000, 151 000, 160 000, and 284 000, although these are not present in microtubules carried through four assembly-disassembly cycles. Evidence is also presented which shows that a 68 000-dalton protein which is a prominent component of HeLa microtubules after two polymerization-depolymerization cycles does not stoichiometrically copurify with tubulin through repeated assembly--disassembly cycles and does not stimulate tubulin polymerization. On the other hand, the sedimentation of this 68 000-dalton protein is apparently influenced by the presence of polymerized microtubules, suggesting that this protein may be a component of a system whjich interacts weakly with microtubules. Finally, evidence is presented suggesting that two-cycle microtubules contain a proteolytic activity that can digest the 201 000-206 000-dalton proteins.
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Rauscher leukaemia virus (RLV), extensively purified by rate and density gradient centrifugation procedures, exhibited aminopeptidase (AP) activity. The amount of virion activity was about 0.005 times the specific activity of purified hog kidney aminopeptidase M (EC 3.4.11.2). The activity was also found in purified Moloney and Gross leukaemia viruses, but not in comparable gradient fractions of uninfected JLSV-9 cells. Furthermore replacement of serum by bovine serum albumin in the growth medium of virus producing cells did not affect the AP activity. These results indicate that murine leukaemia viruses (MuLV) possess a tightly bound AP activity which is present as a minor component of the virion preparation and is probably not due to host cell membrane or serum contamination. Characterization of the pH optimum and substrate specificity show the MuLV aminopeptidase activity is similar to but different from both hog kidney, leucine aminopeptidase (EC 3.4.11.1) and aminopeptidase M (EC 3.4.11.2).
The Pr65gag proteolytic factor obtained from Moloney (MoLV) or Rauscher (RLV) leukaemia virus has been characterized. We found that it was present in small amounts in virions and was extremely unstable. Although it eluted at the trailing edge of p12 on Sephadex G-75 columns, it could clearly be separated from p12 on DEAE-Sephadex A-50M columns, making it unlikely that the factor is p12 or any other major murine leukaemia virus (MuLV) protein. This fact also distinguishes the murine factor from the avian tumour viruses and is stable to column purification methods (von der Helm, 1977; Dittmar & Moelling, 1978). We further observed that: (i) the murine proteolytic factor had an estimated mol. wt. of 20,000 to 22,000, relative to MuLV p12, which eluted as a dimer on Sephadex G-75 columns in the presence of 0.1% NP-40; and (ii) in vitro cleavage of an iodinated Pr65gag-rich, p30-deficient substrate yielded a clear increase in both p30 and p12, which suggests that the in vitro cleavage of Pr65gag is similar to its processing in vivo.
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