Low Molecular Mass Form Research Articles

Protein tyrosine dephosphorylation and signal transduction

The protein tyrosine phosphatases comprise a family of enzymes that specifically dephosphorylate tyrosyl residues. Determination of the amino acid sequence of a major low molecular mass form isolated from human placenta (PTPase 1B) provided the basis for the first identification of transmembrane proteins that bear intracellular phosphatase domains. The existence of such molecules, bearing the hallmarks of receptors, raises the exciting possibility of a novel mechanism of signal transduction in which the early events involve the ligand-induced dephosphorylation of tyrosyl residues in proteins.

Characterization of the Calmodulin-binding and of the Catalytic Domains of Bordetella pertussis Adenylate Cyclase

The structural organization of the low molecular mass form (43 kDa) of Bordetella pertussis adenylate cyclase was dissected taking advantage of the known sequence of the bacterial cya gene (Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2, 19-30) and its low content of Trp and Met residues. Cleavage of the 43-kDa protein and of its complementary tryptic fragments (T25 and T18 peptides) with N-chlorosuccinimide and cyanogen bromide followed by sodium dodecyl sulfate-polyacrylamide gel analysis of digestion products allowed the following conclusions: (i) the catalytically active 43-kDa form of B. pertussis adenylate cyclase is within the first 400 residues of the protein encoded by the cya gene. T25 occupies the N-terminal domain of the protein (residues 1-235/237). Isolated T25 fragment exhibits a low but measurable enzymatic activity which indicates that it harbors the catalytic site; (ii) T18 which is the main calmodulin-binding domain, occupies the C-terminal segment of protein (residues 236/238-399) and is devoid of catalytic properties; (iii) the two complementary peptides T25 and T18 reassociated only in the presence of calmodulin, leading to significant recovery of the original activity. These results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.

Oligomeric states of the insulin receptor: binding and autophosphorylation properties.

Properties of oligomeric states of the insulin receptor were analyzed by polyacrylamide gel electrophoresis in nondenaturing buffer conditions (ND-PAGE). Partially purified insulin receptors resolve in ND-PAGE as three distinct species: (i) the fast electrophoretic mobility, low molecular mass form manifests intense labeling by iodinated insulin and shows basal and insulin-stimulated autophosphorylation; (ii) the middle, intermediate mobility form exhibits strong labeling by iodinated ligand but does not possess the capacity to be autophosphorylated; (iii) the slow mobility, highest molecular mass form necessitates covalent binding with iodinated hormone to withstand electrophoresis and shows autophosphorylation enhanced by insulin. This receptor form is more heavily labeled by phosphorylation than the low form. At 22 degrees C, binding and autophosphorylation do not appear to be time dependent. At 37 degrees C, binding and autophosphorylation of low and high species attain a maximum after 15 min and then decrease as time of incubation with insulin is prolonged to 120 min; the middle species exhibits a much slower association rate, and its labeling by iodinated hormone becomes more intense with time. Our data show that in cell-free systems insulin receptors appear in various oligomeric states and that the highest molecular mass oligomer exhibits the most pronounced autophosphorylation. This is compatible with the concept that insulin receptor oligomerization provides a mechanism for transmembrane signaling.

Serum Form of the Rat Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor Is Truncated in the Carboxyl-terminal Domain

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.

Synthesis and secretion of multiple forms of beta 2-interferon/B-cell differentiation factor 2/hepatocyte-stimulating factor by human fibroblasts and monocytes.

The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2). When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity. We have prepared a rabbit polyclonal antiserum to the E. coli-derived human IFN-beta 2 fusion protein. This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations. The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes. "Uninduced" human FS-4 fibroblasts as well as those induced with interleukin-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30). Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by [3H]glucosamine (gp23-gp25). The inclusion of cycloheximide during the [35S]methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet. Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells. Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.

Two Developmentally Regulated Isoenzymes of Calmodulin‐Stimulated Protein Kinase II in Rat Forebrain

Soluble calmodulin-stimulated protein kinase II has been purified from adult and 10-day-old rat forebrain. By autoradiography, the alpha/beta subunit ratios of the 10-day and adult enzymes were 0.67 +/- 0.03 and 2.20 +/- 0.15, respectively. By silver staining, the alpha/beta subunit ratios were 1.02 +/- 0.06 and 2.36 +/- 0.10, respectively. The apparent holoenzyme molecular masses of the purified 10-day and adult enzymes were 500,000 daltons and 700,000 daltons. However, varying the purification conditions revealed higher and lower molecular mass forms at both ages and suggested that the form of the kinase that is usually purified is merely that which has the highest affinity for calmodulin-Sepharose and may not be the form of the kinase that exists in vivo. The subunits of the adult and 10-day enzymes were indistinguishable by one- and two-dimensional electrophoresis and one-dimensional proteolytic peptide maps. These results are consistent with the suggestion that at least two developmentally regulated isoenzymes of this kinase exist in rat forebrain.

Electrophoretic and Immunochemical Study of the Lipopolysaccharides Produced by Chemostat-grown Escherichia coli 0157

Two chemically different O-polysaccharides, a low molecular mass form of LPS and core LPS produced by chemostat-grown E. coli O157, were analysed by SDS-PAGE, silver staining and immunoblotting. The reactivities of the different O-polysaccharides with antisera prepared against E. coli O157 grown in batch culture, Salmonella O30 or Brucella abortus were very similar, showing that the O-polysaccharides share at least some antigenic determinants. The reactions of the low molecular mass LPS with the antisera indicated it was semi-rough LPS having one repeat unit of the O-polysaccharide attached to core LPS.

The head activator is released from regenerating Hydra bound to a carrier molecule

Hydra forced to regenerate a head releases head activator and head inhibitor during the first hours after cutting to induce head-specific growth and differentiation processes. Analysis of the size distribution demonstrated that the head-activator peptide is co-released with (a) large molecular weight carrier molecule(s) to which it is non-covalently bound. The carrier-bound head activator is fully active on Hydra indicating that a carrier does not hinder the interaction with receptors. In contrast to this the head inhibitor is released in its naked, low molecular mass form. The association or non-association with a carrier molecule results in marked differences in biological properties. The head activator has a short range of action, but a long half-life, the head inhibitor has a global range of action, but a short half-life. These results provide a plausible explanation why two antagonistically acting substances, although they are released from the same site and simultaneously nevertheless can give rise to a well-defined temporal and spatial pattern of differentiation as occurs, for example, during head regeneration in Hydra.

Copper and iron complexes catalytic for oxygen radical reactions in sweat from human athletes

Sweat collected from ‘explosive’ and ‘endurance’ athletes immediately after exercise contains low molecular mass complexes of copper detectable by their ability to bind to o-phenanthroline. Concentrations of these copper complexes are much greater in arm sweat than in trunk sweat. The iron content of arm sweat, as determined by the ferrozine method, is also greater than that of trunk sweat. However, much of the iron in trunk sweat exists in a low molecular mass form that can bind to the antibiotic bleomycin, whereas the iron in arm sweat does not exist in this form. The metal complexes in human sweat are capable of stimulating the peroxidation of membrane lipids in the presence of ascorbic acid. The physiological significance of the presence of iron and copper complexes in sweat is discussed.

Human placental beta-galactosidase: structural and immunological observations.

beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

Mitochondrial creatine kinase (EC 2.7.3.2) in the brain

No CK MM was found in human brain tissue. Both human and rat brain tissue, however, contain a non MM, non BB CK isozyme. The protein is membrane bound. Evidence is presented that it is a mitochondrial variant of the enzyme. This mitochondrial brain CK occurs in two forms. Both a low molecular mass form ( M r = 65000) and a high molecular mass form are detectable. The isoelectric point of the low molecular mass form, Br-Ck ml, is similar to the isoelectric point of CK MM. Complications may arise when techniques are used that try to separate the mitochondrial CK forms from CK MM on basis of differences in net charge. The two mitochondrial brain CK forms are called Br-CK m1 and Br-CK m2. Whether these two enzymes are identical to the mitochondrial heart CK forms remains unclear.

Inactivation and reactivation of rat liver 3-hydroxy-3-methylglutaryl-CoA-reductase phosphatases: effect of phosphate, pyrophosphate and divalent cations.

Incubation of four purified rat liver HMG-CoA-reductase phosphatases (Gil, G., Sitges, M. and Hegardt, F.G. (1981) Biochim. Biophys. Acta 663, 211-221) with Mn2 or Mg2 caused a concentration-dependent activation of enzyme activities. The maximum effect for Mn2 was at 5 mM for all phosphatases. Fe2 caused inactivation only in reductase phosphatases IIa and IIb. Ca2 10 mM showed a slight effect of inactivation. Phosphate, pyrophosphate and adenine nucleotides inhibited the four reductase phosphatases, this process being concentration-dependent. cAMP did not inhibit the four phosphatases at all in the range of 0.01-8 mM. Preincubation of reductase phosphatases with PPi and subsequent dilution did not diminish the inactivation effect, showing that this ion inhibits the enzyme prior to the binding to the substrate. Phosphorylated sugars, but not free sugar, inactivated the four reductase phosphatases. PPi-inactivated enzymes were reactivated by Mg2 or Mn2, this process being time-dependent. The four phosphatases had different patterns of reactivation. Phosphatases Ib and IIb (low-molecular mass forms) were shown to be different enzymes as judged by: their divergent behaviour when inhibited with Fe2; their PPi response; kinetics of reactivation by Mg2 or Mn2 or PPi-inactivated enzymes; and thermal stability. A metalloenzyme character is suggested for reductase phosphatases.

Immunochemical characterization of human plasminogen activators

The high and low molecular mass forms of human urokinase (high M r urokinase and low M r urokinase) were purified to homogeneity and the human vascular plasminogen activator was partially purified. The urokinase molecules were radiolabelled with 125-iodine and assays of radioimmune binding and inhibition of binding were carried out. The high M r form was almost as good an inhibitor as the low M r form in the binding reaction between radiolabelled low M r urokinase and specific antibody, indicating that the low M r urokinase is a part of the high M r urokinase. This inhibition could be only partly reversed, indicating that the high M r urokinase has immunological determinants which are not present on the low M r urokinase. The human vascular plasminogen activator preparation partly inhibited the urokinase binding reactions. Contaminating proteins could be responsible for this inhibitory effect. Various human sera inhibited the binding reaction between the two forms of urokinase and antibody, indicating that normally either urokinase or an immunologically cross-reacting molecule is present in human sera. The inhibitory capacity of sera from patients with neoplastic disease was not different from that of other sera.