Breast cancer and T-cell-mediated immunity to proteins of the mouse mammary tumour virus (MMTV).

We have previously reported that breast cancer patients and some healthy subjects show positive T-cell-mediated immune responses to a semi-purified mouse mammary tumour viral pool (MMTV). We have now used Western blotting to analyse the specificity of the response and to determine the target polypeptides. Two types of T-cell response to the viral antigens were examined, proliferation and MIF release, the latter implies a DTH status in vivo where primed lymphocytes are involved. Two viral fractions were used, one containing a glycoprotein, the 52 kD major virus envelope, and the other containing the 28 kD main virus core protein. We analysed both patients and healthy subjects whose T-cells proliferated to the MMTV total extract (viral pool). The T-cell response in the patients was shown to be viral specific since both the T-cell proliferation (21/25) and MIF release (17/19) were directed against viral components of the pool (gp 52 and/or p 28). The T-cell response in the healthy control subjects was shown to be mostly directed against a species-specific albumin component of the extract. In addition, the monocyte integrity required for the MIF response was altered in the breast cancer patients. The monocytes from one patient out of three failed to respond to MIF, even though the lymphokine was released normally by the patients' activated T-cells.

Reactions of nucleic acid bases with alpha-acetylenic esters. Synthesis and enzymatic evaluation of chemically modified nucleotides.

Analogues of adenine nucleotides, containing an additional chloromethyl-pyrimidone ring fused to the purine base, were obtained by treatment of AMP, ADP and ATP with an alpha-acetylenic ester, methyl 4-chlorobut-2-ynoate. These compounds were tested for their ability to substitute for the natural substrates or cofactors of several enzymes. With the ADP analogue, pyruvate kinase showed a significant increase of the Km value and a moderate decrease of V, while the reverse was observed when hexokinase was tested with modified ATP. Adenylate kinase was active with the ATP analogue but not with the AMP derivative. Myosin accepted the ATP analogue as a substrate, but was irreversibly modified. Among the dehydrogenases tested, only glucose-6-phosphate dehydrogenase was inhibited by the nucleotide analogue. The structure--activity relationship of these nucleotide derivatives, which represent the largest dimensional deviation known from natural nucleotides, is discussed in comparison with some earlier described dimensional probes of enzyme-nucleotide binding sites.

Effects of serum vitamin-D-binding protein on actin in the presence of plasma gelsolin.

The interaction of serum vitamin-D-binding protein (DBP) and plasma gelsolin with actin was studied using fluorescent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-actin or N-pyrenylcarboxyamidomethyl-actin. DBP and gelsolin formed very tight complexes with one or two monomeric actin subunits respectively. Kd values of about 10 nm have been found for both complexes. When DBP and gelsolin were added simultaneously to G-actin no ternary complex was observed and the DBP-actin complex was formed preferentially. In the presence of CaCl2 no transfer of actin occurred from the 1:2 gelsolin:actin molar complex to free DBP while in the presence of EGTA one actin monomer could be transferred. DBP did not affect the severing activity of gelsolin. The effects of a mixture of gelsolin and DBP on F-actin suggest that only individual interactions of these two plasma proteins with actin occurred in solution.

Cytoskeletons of ADP- and thrombin-stimulated blood platelets. Presence of a caldesmon-like protein, alpha-actinin and gelsolin at different steps of the stimulation.

Comparative analyses of the cytoskeletons of resting and stimulated platelets point out the involvement of a 79 kDa polypeptide in the activation step and its increased incorporation during aggregation. It appears as a doublet and cross-reacts with an antibody to chicken gizzard caldesmon, whereas no 150 kDa immunoreactive form was detected. alpha-Actinin and gelsolin were detected only in the aggregation step.

Properties of two isoforms of human blood platelet alpha-actinin.

The structural and functional properties of the aa (2 X 97 kDa) and cc (2 X 94 kDa) isoforms of platelet alpha-actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross-reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F-actin (high-speed sedimentation) and cross-link or gel actin filaments (low-speed sedimentation and viscometry), in a calcium-dependent manner. The study of the interaction with F-actin indicates that the binding of 1 molecule of aa or cc alpha-actinin/9-11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCl2 at 0.1 mM strongly inhibits the binding of aa to F-actin and weakly that of cc, while it inhibits similarly the cross-linking of either aa or cc. The cross-linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of aa at 4, 20, 30 and 37 degrees C, respectively. The bb form (2 X 96 kDa), which is a proteolytic product of aa [Y. Gache et al. (1984) Biochem. Biophys. Res. Commun. 124, 877-881], behaves roughly as aa, but the calcium sensitivity of its binding to F-actin is intermediate between that of aa and cc. These results suggest that the effect of Ca2+ concentration on the binding of platelet alpha-actinin to F-actin may be partly dissociated from the effect on the cross-linking.

Bromopyruvate, a potential affinity label for octopine dehydrogenase.

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.

Susceptibility of platelet alpha-actinin to a Ca2+-activated neutral protease.

Purified alpha-actinin from human platelets was digested with Ca2+-activated protease from muscle. The alpha subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, alpha-actinin is cleaved by the endogenous Ca2+-activated protease.

Isolation and characterization of human blood platelet gelsolin.

A 90-kDa protein-actin stable complex was purified from blood platelets by a short and efficient procedure giving at the same time actin used in polymerization assays. 90-kDa protein free of actin was prepared from the complex by 8 M ureau treatment and renaturation. By its molecular mass, immunological cross-reactivity with macrophage gelsolin and its effect on G- and F-actin the 90-kDa protein appears as the platelet gelsolin.

Evidence for the presence of tropomyosin in the cytoskeletons of ADP- and thrombin-stimulated blood platelets.

Stimulation of porcine platelets with ADP or thrombin and subsequent analyses of their cytoskeletons by SDS-polyacrylamide gel electrophoresis have shown the presence of a 30.5-kDa polypeptide in the cytoskeletons of activated as well as aggregated platelets. This polypeptide comigrates with pure porcine platelet tropomyosin in SDS gels, their mobilities being similarly and markedly decreased in the presence of 6 M urea. One-dimensional peptide mapping after limited proteolysis by Staphylococcus aureus protease gives the same pattern for pure tropomyosin and the 30.5-kDa polypeptide. This latter may thus be identified as the porcine platelet tropomyosin subunit, the role of which may not be solely structural.

Polymorphism of alpha-actinin from human blood platelets. Homodimeric and heterodimeric forms.

In purified solutions of alpha-actinin from human blood platelets, three polypeptides a, b and c, of approximately 100 kDa, were observed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were identified as alpha-actinin subunits on the basis of their cross-reactivity with antibodies against skeletal muscle alpha-actinin and their interaction with F-actin. After electrophoresis in the absence of sodium dodecyl sulfate, six forms were observed: aa, ab, bb, ac, bc, cc. The similarity between the one-dimensional peptide maps of a and b and the absence of b in the presence of calcium-dependent protease inhibitors indicated that b is probably derived from the a subunit. The c subunit differs from the a subunit. The results provide evidence that there are actually only two platelet alpha-actinin subunits a and c which give rise to three isoforms: two homodimers aa and cc, and one heterodimer ac.

The effect of serum vitamin D-binding protein on polymerization and depolymerization of actin is similar to the effect of profilin on actin.

The mechanism of the interaction between two genetically determined serum vitamin D-binding protein forms and the muscle skeletal actin was investigated. Vitamin D-binding protein was isolated in a good yield from human serum, using immunoaffinity chromatography. 16 mg of pure vitamin D-binding protein were obtained from 100 ml of serum. The interaction between purified vitamin D-binding protein and skeletal muscle actin was studied by viscosity, delta A (232 nm) measurements and by electron microscopy. The effect of vitamin D-binding protein on actin polymerization is characterized by the decrease of the nucleation and elongation rates and by the decrease of the final concentration of polymerized actin in the steady state. The depolymerizing effect is not the result of direct action on vitamin D-binding protein on F-actin but rather of an increased concentration of the complex of the former protein with G-actin. The characteristics of the vitamin D-binding protein and profilin interactions with actin are similar. Both proteins seem to react only with G-actin.

Isolation and physico-chemical properties of blood platelet alpha-actinin.

A procedure for the isolation of alpha-actinin from human blood platelets is described. Typical yields were 10-13 mg from 48 g of frozen platelets. The purified platelet alpha-actinin has many physico-chemical properties (molecular weight in native state, molecular weight in denaturing conditions, Stokes radius, ellipticities at 208 and 221 nm) similar to those of muscle alpha-actinins. However, in contrast to muscle alpha-actinins, it is composed of isoforms containing subunits of slightly different molecular weights and its effect on actin gelation is calcium-sensitive. These two characteristics are common to other known non-muscle alpha-actinins.

Comparison of the properties of two kinds of preparations of human blood platelet actin with sarcomeric actin.

A new procedure of purification of actin from human blood platelets was used. This method starting from acetone powder of whole platelets gives a much higher yield than the one previously described (actin I) (Landon et al. (1977) Eur. J. Biochem., 81, 571-577). This actin II preparation has the same reduced viscosity as skeletal muscle actin, while the reduced viscosity of actin I preparation is about 1/10 of this value. Moreover actin I has the form of very short filaments as shown by electron microscopy. After an extra step of purification actin I, when polymerized, acquired a high reduced viscosity. We confirmed that platelet and sarcomeric actins are similar in their polymerization properties and their ability to activate muscular myosin. A circular dichroism study showed that the overall conformation of both actins are similar, but the environment of their aromatic chromophores is different.

Muscle beta-actinin and serum albumin of the chicken are indistinguishable by physicochemical and immunological criteria.

Chicken muscle beta-actinin is considered to be one of the "true" myofibrillar components due to its specific binding to isolated myofibrils. Surprisingly, the direct comparison of this muscle protein with serum albumin, both isolated from chicken, showed that they behaved identically under several electrophoretic conditions. Furthermore, immunoreplica gels and double-immunodiffusion tests with antibodies prepared against beta-actinin established the serological identity of both proteins. No significant differences were found by circular dichroic spectroscopy or in amino acid composition. In addition, the amino-terminal sequences of both proteins were identical (H2N-Asp-Ala-Glu-His-Lys-Ser-Glu-Ile-Ala-His-Arg-Tyr-Asn-Asp-Leu-). Combined, these results strongly indicate that muscle beta-actinin and serum albumin are similar, if not identical.

Isolation and characterization of two molecular forms of octopine dehydrogenase from Pecten maximus L.

Two forms, A and B, of octopine dehydrogenase from Pecten muximus L. straited adductor muscles were separated by ion-exchange chromatography and purified to homogeneity. Their kinetic properties were similar and, among others, the mnemonical behaviour, previously found for octopine dehydrogenase, was confirmed. Structural features, determined by polyacrylamide gel electrophoresis with and without sodium dodecylsulfate, amino acid composition, immunological tests and heat stability were compared. The only differences between the two forms are their charge and their sensibility to various chemical treatments. Assays of reciprocal conversion of the two forms by oxidation, reduction or deamidation failed. No tissue specificity and no relation to any physiological conditions could be observed. However, a constant ratio A:B = 1:4 was statistically found in crude extracts as well as in the purified enzyme. It therefore seems possible to assume that the two forms of octopine dehydrogenase pre-exist in living P. maximus, although their genetic origin has to be established.

Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.

The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine.

Fluorescence properties of reduced thionicotinamide--adenine dinucleotide and of its complex with octopine dehydrogenase.

Reduced 3-thionicotinamide--adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 +/- 0.5) x 10(-4). The interaction between sNADH and octopine dehydrogenase was investigated by ultraviolet-difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine, as follows. (a) There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40-nm blue shift of the emission maximum. (b) The protein fluorescence is quenched almost completely. (c) The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring the free form. These features appear to be unique to the octopine.sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. It was found that sNAD neither binds nor acts kinetically as a coenzyme. sNADH exhibits relatively good binding, with Km and Ki values close to those of the natural coenzyme, but the turnover number is 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH dissociation constants were determined as a function of temperature, and appear to be practically temperature-independent in the range 10--40 degrees C. It seems thus, in agreement with previous studies, that the interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity, and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed.

Human platelet actin. Evidence of beta and gamma forms and similarity of properties with sarcomeric actin.

Human blood platelet actin was purified using 30% sucrose to extract actomyosin and potassium iodide to dissociate actomyosin and to depolymerize actin. Pure actin thus obtained resembles skeletic muscle actin in its polymerization properties, CD spectra and ability to activate myosin myosin Mg2+-ATPase. Isoelectric focusing gel analysis shows that human blood platelet actin exists in beta and gamma forms. The ratio of beta to gamma forms is of 5 in purified actin, in whole cell extract and in all the fractions studied.

Pulse fluorimetry study of octopine dehydrogenase-reduced nicotinamide adenine dinucleotide complexes.

We measured the transient fluorescence of NADH bound to octopine dehydrogenase in the binary octopine dehydrogenase-NADH complex and in the ternary complexes containing D-octopine, L-allooctopine, L-arginine, D-arginine, or 5-guanidinovaleric acid. The fluorescence decay in all these complexes is biexponential. This is explained by the presence of several conformations of the single NADH binding site. In addition, transietn anisotropy measurements show that the nicotinamide moiety is rigidly bound to the enzyme.