Identification and characterisation of an antithrombin III mutant (AT Dublin 2) with marginally decreased heparin reactivity.

The antithrombin III (ATIII) isoform patterns of plasma and serum samples from cancer patients and controls were analysed by isoelectric focusing and immunoblotting. A novel ATIII banding pattern was identified in two individuals: a patient with breast carcinoma who developed deep venous thrombosis and a blood donor. Family studies in the patient showed the abnormal pattern to be due to a mutant form of ATIII (AT Dublin 2). The coagulation properties of AT Dublin 2 heterozygotes were normal. Immunologic and activity levels of ATIII, measured by standard techniques, were normal. Mutant plasma ATIII showed reduced thrombin reactivity at low concentrations of thrombin and demonstrated decreased reactivity with heparin over a range of heparin concentrations. This was confirmed using a modified ATIII heparin cofactor activity assay with varying heparin concentrations. The abnormal ATIII was also found to elute from heparin agarose at a lower ionic strength than normal ATIII. Two dimensional gel electrophoresis showed the abnormal ATIII to have similar molecular size distribution to normal ATIII. Neuraminidase treatment of normal and mutant plasma reduced the ATIII isoforms to one in both samples. The possible role of AT Dublin 2 in predisposing to hypercoagulation is discussed.

Identification and characterization of a new antithrombin III familial variant (AT Dublin) with possible increased frequency in children with cancer.

The antithrombin III (ATIII) isoform pattern of a number of serum and plasma samples was analysed by isoelectric focusing and immuno-blotting. A novel ATIII isoform pattern which was observed in 4/80 children with acute lymphatic leukaemia (ALL) and in 1/4 children with Ewing's sarcoma, has been shown by family studies to be due to a mutant form of ATIII (AT Dublin) in the heterozygous state. The coagulation properties of AT Dublin heterozygotes were normal. In addition the immunological and activity levels of their ATIII were normal. The effects of thrombin and heparin on the mutant ATIII were similar to controls. Neuraminidase treatment reduced the ATIII isoforms to one in controls and two in the mutant. Two-dimensional gel analysis showed the mutant ATIII to have an identical molecular size distribution to the normal form. This mutant is, thus, most likely due to an amino acid substitution giving a more basic molecule that is clinically silent (at the coagulation level). It may be of interest that the frequency of AT Dublin in the ALL group is significantly higher than in the control group (3/430) studied (P less than 0.001).

Inhibition of reverse transcriptase by polyvinyl sulfate (PVS).

The effect of the ribonuclease inhibitor polyvinyl sulfate on the activity of retroviral DNA polymerase and terminal c transferase was examined. This substance was found to be a potent inhibitor of these enzymes by virtue of competition between the sulfated sidechains of the molecule and the template primer for a site on the enzyme. The significance of these findings in relation to searching for reverse transcriptase is discussed.

Isolation and characterisation of the L cell virion DNA polymerase.

The DNA polymerase of the L-cell virion (LCV) was partially purified by chromatography on DEAE cellulose. This enzyme transcribed poly(A) . oligo (dT), poly(C) . oligo(dG), and poly(Cm) . oligo(dG), had a molecular weight of 77,000 daltons and reacted like other murine viral RNA directed DNA polymerases to anti reverse transcriptase specific IgG preparations indicating that it was probably a typical murine viral reverse transcriptase. In addition, like other partially purified mammalian viral reverse transcriptases the LCV DNA polymerase exhibited template independent primer stimulated DNA synthesis. The significance of these results to the unusual endogenous activity of the LCV is discussed.

Demonstration of an unusual DNA polymerase activity associated with the L cell virion.

Purified preparations of L cell virions (LCV) were found to possess an associated DNA polymerase activity. This enzyme was active with poly(C).oligo(dG) and poly(Cm).oligo(dG) and was able to transcribe poly(A).oligo(dT). Endogenous DNA synthesis was also demonstrable in disrupted virion preparations but this reaction was enhanced, rather than inhibited, by RNase pre-treatment. The effects of variations in a number of the assay parameters on these activities were examined in an attempt to determine the class of DNA polymerase involved.

Studies of the template preference and other characteristics of the DNA polymerases of leukemic guinea pig lymphoblasts.

A single peak of DNA polymerase activity was detectable by phosphocellulose chromatography of leukemic guinea pig lymphoblast whole cell extracts. The inability to detect multiple peaks of activity as described with other cell types is shown to be due to the insolubility of a large proportion of the DNA polymerase activity under the extraction condition used. Multiple forms of DNA polymerase with different template specificities were recognized in extracts of the subcellular fractions of these cells after chromatography on phosphocellulose and DEAE cellulose. On Sephadex G-200 gel filtration these enzymes had apparent molecular weights in excess of 140,000 daltons. No RNA polymerase (reverse transcriptase) was detected in any subcellular fraction despite the presence of oncornavirus like particles in these cells.

DNA polymerase activity in plasma from leukaemic guinea-pigs.

Measurement of DNA polymerase in leukaemic guinea-pig plasms reveals the presence of low levels of sedimentable and non-sedimentable enzymic activities. Since the sedimentable DNA polymerase is ribonuclease sensitive, uses poly(C).oligo(dG) as template, and bands in a sucrose density gradient at 1-17 g/ml it is thought to be the GPLV-associated reverse transcriptase. The soluble DNA polymerase is stimulated by ribonuclease and is probably of cellular origin.