Mammalian high molecular weight and monomeric forms of valyl-tRNA synthetase

Valyl-tRNA synthetase from rat liver sediments at 15.5 S with a Stokes radius of 90 A, corresponding to a native molecular weight of 585,000. Purification of valyl-tRNA synthetase to homogeneity by a combination of conventional and affinity column chromatography yields a fully active monomeric form of valyl-tRNA synthetase with a sedimentation coefficient of 7.7 S and a Stokes radius of 45 A. The subunit molecular weight of the monomeric valyl-tRNA synthetase is 140,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the presence of 400 mM KCl, the purified monomeric valyl-tRNA synthetase associates to a high molecular weight form. The high molecular weight valyl-tRNA synthetase in the homogenate can be readily converted to the monomeric form by controlled trypsinization. The kinetic parameters of the two forms are nearly identical. The results suggest that the high molecular weight valyl-tRNA synthetase is a homotypic tetramer and converts to the monomeric valyl-tRNA synthetase after the cleavage of a small peptide.     

Steroidogenic activity of high molecular weight forms of corticotropin.

The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Calcium-activated potassium transport and high molecular weight forms of calpromotin.

Investigations of human red blood cells show that a cytoplasmic protein called calpromotin is involved in the regulation of calcium-activated potassium transport. Calpromotin associates with the membrane in the presence of calcium and undergoes a chemical transformation. High performance gel filtration and gel electrophoresis show that the cytoplasmic and membrane-bound calpromotin can exist in both low and high molecular weight forms. The biochemical properties of the high molecular weight membrane-bound calpromotin are not the same as the high molecular weight cytoplasmic calpromotin. The high molecular weight membrane forms of calpromotin are increased by leupeptin and diminished by iodoacetic acid. Therefore, the leupeptin enhancement and iodoacetic inhibition of calcium-activated potassium transport may involve the high molecular weight forms of membrane-bound calpromotin.     

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

High molecular weight forms of GTP-binding regulatory proteins from the bovine cerebellum

A new form of a low Km GTPase belonging to the family of regulatory GTP-binding G-proteins has been identified in bovine cerebellum. The molecular weight of this G-protein is several times as high as that of other G-proteins known to be alpha beta gamma heterotrimers: i. e., Gs, Gi, Go, transducin and a new G-protein which had recently been isolated in our laboratory from bovine cerebellum. The high molecular weight G-protein is stable against dissociation; its molecular mass does not change after treatment with DTT, colchicine and NaF. Using antibodies against the alpha-subunit of the formerly isolated cerebellar G-protein and the transducin beta-subunit, it was demonstrated that the both immunoreactive subunits are present in the high molecular weight G-protein. The two forms of the cerebellar G-proteins, i. e., "high" and "low molecular weight" ones, differ drastically in terms of the Mg2+ effect on their GTPase activity. Whereas at submicromolar concentrations of Mg2+ the GTPase activity of the former is virtually absent, the GTPase activity of the latter is more elevated in the presence of EDTA than in the presence of Mg2+.     

Localization of major, high molecular weight proteins in Bacteroides forsythus

Bacteroides forsythus produces species-specific major proteins with high molecular weights of 270 and 230-kDa (270K and 230K). A specific antibody raised against 270K was used for Western blot analysis and immunoelectron microscopy. Western blot analysis showed that the 270K and 230K proteins were immunologically similar. Immunogold labeling of ultrathin-sectioned bacterial cells and biochemical fractionation revealed that these proteins were localized at the outermost cell surface, not in the cytoplasm. These results suggest that major proteins ubiquitous to this species may form the S-layer.     

Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60^c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60^c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60^c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt - CNS central nervous system - DPBS Dulbecco's phosphate-buffered saline - GAM-AP goat anti-mouse IgG alkaline phosphatase conjugate - LB limb bud - NF high molecular weight neurofilament protein - NBT nitroblue tetrazolium chloride - SDS-PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - RIPA radioimmunoprecipitation - TBS Tris-buffered saline - TTBS TBS with 0.05% Tween-20 Presented in part at the 1989 and 1990 Teratology Society Meetings and the 1990 Society of Toxicology Meetings.     

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.     

Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments

The mammalian neurofilament triplet proteins (210, 160 and 68 × 10³M_r proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 10³M_r protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 10³M_r protein to 68 × 10₃M_r protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 10₃M_r protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.     

Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor

Tumor necrosis factor (TNF) is synthesized as a prohormone with an unusually long and atypical signal sequence which is absent from the mature secreted cytokine. In addition to mature 17 kDa TNF, LPS-stimulated murine macrophages secrete at least seven TNF-like proteins (isoforms) of differing electrophoretic mobility which appear as a "ladder" on SDS-PAGE. We here present data indicating that these isoforms derive not from sequential clipping of propiece fragments, but rather from differential glycosylation at sites on the mature hormone. Selected isoforms have been isolated and purified by sequential chromatographic and electrophoretic steps. NH2-terminal sequence analysis of two of these isoforms reveal sequences identical to that of mature 17 kDa murine TNF. Characterization of the secretory products of tunicamycin-treated. LPS-stimulated murine macrophages indicate that the "ladder" complex reflects differential glycosylation of mature 17 kDa TNF. Digestion of purified isoforms with a battery of glycosidic enzymes indicate that secreted forms of murine TNF contain both sialic acid and asparagine(N)-linked chains. The biological significance of this heterogeneity is not known.     

Mapping of caldesmon: relationship between the high and low molecular weight forms

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.     

The high molecular weight proteins released from cultured cells.

Studies have been performed with the serum-free culture medium taken from several fibroblast monolayer culture lines. A high molecular weight protein fraction was separated from the concentrated medium by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis was used to assess the degree of purification obtained. In the electron microscope the negatively stained high molecular weight proteins were found to closely resemble the alpha2-macroglobulins. The suggestion that these proteins from cultured cells resemble the cylindrical protein complex isolated from mammalian erythrocyte ghosts is not supported by this study. The results are discussed in the light of the extensive literature now available on the electron microscopy of high molecular weight proteins.     

Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.     

High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal.

Protein adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimer's disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1-33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.     

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…     

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.