High Molecular Weight Polypeptides Research Articles

N-Terminus Conservation in the Terminal Pigment of Phycobilisomes from a Prokaryotic and Eukaryotic Alga

High molecular weight polypeptides from phycobilisomes, believed to be involved in facilitating the energy flow from phycobilisomes to thylakoids, are conserved in the prokaryote Nostoc sp. and the eukaryote Porphyridium cruentum. Partial N-terminal sequence analysis of the phycobilisome-polypeptides of Nostoc (94 kilodalton) and Porphyridium (92 kilodalton) revealed 55% identity in the first 20 residues, but no significant homology with sequences of other phycobiliproteins or phycobilisome-linkers. Polypeptides (94 and 92 kilodalton) from Nostoc thylakoids free of phycobilisomes, previously presumed to be involved in the phycobilisome-thylakoid linkage (M Mimuro, CA Lipschultz, E Gantt 1986 Biochim Biophys Acta 852: 126) exhibit the same immunocrossreactivity but are different from the 94 kilodalton-phycobilisome polypeptide by having blocked N-termini and a different amino acid composition.

Reconstitution of purified cardiac muscle calcium release channel (ryanodine receptor) in planar bilayers

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).

FRACTIONATION OF HIGH MOLECULAR WEIGHT POLYPEPTIDES FROM BEER USING TWO DIMENSIONAL GEL ELECTROPHORESIS

High molecular weight polypeptides from beer were fractionated by a two dimensional gel electrophoretic method comprising isoelectric focusing in the first dimension followed by gradient gel electrophoresis in the second dimension. Silver stained gels revealed a complex pattern of spots which is consistent with individual polypeptide species having both discrete isoelectric points and molecular weights. The pattern of silver stained spots was reproducible both within and between beers and was independent of the sample preparation method or the composition of the sample solubilisation mix. Beers produced from grists containing malt plus 20% wheat flour were shown to contain four polypeptides of approximate molecular weight 30 000, 30 000, 16 000 and 15 000 which were absent from all malt control beers.

Detection of polypeptides associated with the cold acclimation process in spinach

Exposure of spinach (Spinacia oleracea) seedlings to 5 degrees C for several days has previously been shown to induce a greater tolerance to the stresses of extracellular freezing. Associated with this response to low temperature, termed cold acclimation, was a subtle shift in protein synthesis and altered polypeptide composition. Two-dimensional gel electrophoresis was used to study the changes in spinach leaf tissue protein synthesis in an effort to identify polypeptides that may play a central role in the induction of greater freezing tolerance. Through a combination of silver staining, in vivo labeling, and in vitro translation of mRNAs, we identified several high molecular weight polypeptides whose synthesis and presence in spinach leaf tissue were highly correlated with freezing tolerance. Synthesis of these polypeptides was elevated or induced during cold acclimation when freezing tolerance increases, but was rapidly reduced or halted during deacclimation when freezing tolerance declines. The close association of the synthesis of these polypeptides with the induction and loss of freezing tolerance suggested that they could play a role in cold tolerance mechanisms of spinach.

Purified ryanodine receptor of skeletal muscle sarcoplasmic reticulum forms Ca2+-activated oligomeric Ca2+ channels in planar bilayers.

The ryanodine receptor of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle has been purified and found by electron microscopy to be equivalent to the feet structures that are involved in situ in the junctional association of transverse tubules with terminal cisternae of SR. We now find that when the purified receptor is incorporated into vesicle-derived planar bilayers, it forms Ca2+-specific channels, which are dependent on submicromolar Ca2+ for activity. In the presence of 1 mM ATP, the channel shows essentially no activity at 10 nM Ca2+ but becomes highly activated at 50 nM Ca2+. At suboptimal Ca2+ levels (100 nM), the channel is strongly activated by 1 mM ATP and can be blocked by ruthenium red, both effects being prevented by higher Ca2+ levels (1 microM). Mg2+, added from the cis side at millimolar concentrations, blocks Ca2+ flux through the channel from trans to cis (equivalent to flux from luminal to myoplasmic compartment). Ryanodine stabilizes the open state of the channel and blocks the action of ruthenium red to close the channel. Thus, the purified ryanodine receptor incorporated into a bilayer has the Ca2+-channel characteristics consistent with the calcium release observed in isolated terminal cisternae vesicles. Furthermore, ryanodine induced the appearance of a sublevel gating mode characterized by long open conductance states, which were integral multiples of the smallest observed conductance, 3.8 pS in 50 mM Ca2+. The purified receptor consists essentially of a single-sized high molecular weight polypeptide (Mr. approximately equal to 360,000), which on reconstitution forms the square rectangles diagnostic of the feet structures. We conclude that the identity of the Ca2+-release channel of SR is the foot structure, which consists of an oligomer of the high molecular weight polypeptide.

Variation in the precursor to the major merozoite surface antigens of Plasmodium falciparum

The major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight polypeptide. The precursor to the major merozoite surface antigen (PMMSA) has conserved and variable antigenic determinants and varies in size in different isolates. Since the protein is a candidate for a malaria vaccine, it is important to understand the molecular basis of this variation. We present the complete sequence of the PMMSA of the Papua New Guinea isolate FC27 and the partial sequence of the West African isolate NF7. The gene consists of blocks of sequence which are either conserved or variable between different isolates. Variable sequences fall into two distinct types, indicating that the PMMSA is encoded by dimorphic alleles that undergo recombination within conserved blocks at the 5′ end of the gene. Genetic exchange is not apparent within the other two-thirds of the gene in 12 isolates, suggesting strong selection against such recombinants. The most variable block located near the 5′ end contains repeats that are different in independent cloned isolates. This variation presumably accounts for much of the size and antigenic variability.

Mitogen stimulation promotes human T lymphocyte adhesion to fibronectin.

Purified human peripheral blood T lymphocytes were shown to adhere to growth substrata coated with purified human plasma fibronectin (pFn) and its Mr 120,000-140,000 proteolytic fragments containing the cell-binding site. In contrast, significant binding to laminin- or type I collagen-coated surfaces could not be demonstrated. Binding of T cells to pFn could be inhibited by the synthetic peptide Arg-Gly-Asp-Ser. Activation of T lymphocytes with concanavalin A (Con A) and a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), led to a higher adherence to pFn than in unstimulated, resting T cells. Activation with only Con A in the presence of accessory cells also promoted binding. Increased adherence of T cells to pFn could be demonstrated as early as 2 h after the onset of stimulation and reached its maximum in 2-3 days. Furthermore, activated but not resting T cells actively spread on pFn-coated surfaces and displayed an altered F-actin organization. In an overlay assay of electrophoretically separated polypeptides of activated T lymphocytes, pFn bound to a high molecular weight polypeptide of Mr 190,000, suggesting that the cells bind to pFn via a receptor-like molecule. Thus, adhesion ot pFn may be a two-stage process. At the first stage cells bind to Fn. Activated T cells proceed to the second stage, where cells begin to spread on pFn. This may be due to an altered relationship between Fn receptors and microfilaments.

RAPID METHODS FOR DETERMINING THE HIGH MOLECULAR WEIGHT POLYPEPTIDE COMPONENTS OF BEER

Size exclusion chromatography and a coomassie blue dye-binding assay were used to investigate the high molecular weight polypeptide fractions of beer. Size exclusion chromatography using small colums of Sephadex G25, G50 (10 ml) and G75 (20 ml) together with an automated FPLC system enabled the rapid isolation of high molecular weight polypeptide fractions of beer. Size exclusion chromatography using small columns of Sephadex was investigated as a method for the determination of the total high molecular weight polypeptides in beer. The results from the analysis of a series of pilot brewery beers produced under standardised brewing conditions showed that the head retention value of a beer correlated with the content of high molecular weight polypeptide material regardless of the molecular weight fraction assayed. Both methods may be used to monitor the high molecular weight polypeptide content, predict the head retention value of a beer and are sufficiently rapid for routine use in the quality control laboratory.

Vitellogenin in the camel cricket Ceuthophilus sp. (orthoptera, gryllacrididae)

AbstractHemolymph and egg yolk proteins in the camel cricket, Ceuthophilus sp., were analyzed by native and SDS‐polyacrylamide gel electrophoresis. Four sex‐specific polypeptides (FSYPs I–IV) accounted for 44% of the hemolymph protein in adult females. These polypeptides were absent from the blood of adult males and female nymphs but composed 63% of the yolk protein in fully grown oocytes. Hemolymph FSYPs became labeled with [35S]‐methionine after a 1‐hour period of in vivo incorporation and appeared in terminal, vitellogenic follicles after 2 hours of incorporation. Two‐dimensional gel electrophoresis showed that all four FSYPs occur in a single egg‐yolk glycoprotein also present in adult female hemolymph. We conclude that this glycoprotein is a multisubunit vitellogenin (VG). Two high molecular weight polypeptides synthesized by the fat body of adult females and immunoreactive with VG‐specific antiserum were tentatively identified as proVGs.

Identification of a novel 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide in calcium channel preparations.

A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm.

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm × 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm × 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.

Skelemins: cytoskeletal proteins located at the periphery of M-discs in mammalian striated muscle.

The cytoskeletons of mammalian striated and smooth muscles contain a pair of high molecular weight (HMW) polypeptides of 220,000 and 200,000 mol wt, each with isoelectric points of about 5 (Price, M. G., 1984, Am. J. Physiol., 246:H566-572) in a molar ratio of 1:1:20 with desmin. The HMW polypeptides of mammalian muscle have been named "skelemins," because they are in the insoluble cytoskeletons of striated muscle and are at the M-discs. I have used two-dimensional peptide mapping to show that the two skelemin polypeptides are closely related to each another. Polyclonal antibodies directed against skelemins were used to demonstrate that they are immunologically distinct from talin, fodrin, myosin heavy chain, synemin, microtubule-associated proteins, and numerous other proteins of similar molecular weight, and are not oligomers of other muscle proteins. Skelemins appear not to be proteolytic products of larger proteins, as shown by immunoautoradiography on 3% polyacrylamide gels. Skelemins are predominantly cytoskeletal, with little extractable from myofibrils by various salt solutions. Human, bovine, and rat cardiac, skeletal, and smooth muscles, but not chicken muscles, contain proteins cross-reacting with anti-skelemin antibodies. Skelemins are localized by immunofluorescence at the M-lines of cardiac and skeletal muscle, in 0.4-micron-wide smooth striations. Cross sections reveal that skelemins are located at the periphery of the M-discs. Skelemins are seen in threads linking isolated myofibrils at the M-discs. There is sufficient skelemin in striated muscle to wrap around the M-disc about three times, if the skelemin molecules are laid end to end, assuming a length-to-weight ratio similar to M-line protein and other elongated proteins. The results indicate that skelemins form linked rings around the periphery of the myofibrillar M-discs. These cytoskeletal rings may play a role in the maintenance of the structural integrity of striated muscle throughout cycles of contraction and relaxation.

Target antigens of malaria transmission blocking immunity exist as a stable membrane bound complex.

Monoclonal antibodies (MoAbs) blocking transmission of malaria by blocking fertilization of female gametes by male gametes in the mosquito midgut immunoprecipitate three polypeptides (apparent non-reduced mol. wt of 230,000, 48,000, 45,000) from the surface of gametes and zygotes of Plasmodium gallinaceum and P. falciparum. Earlier studies have shown that the epitopes with which the MoAbs react are present only on the mol. wt 230,000 polypeptide in P. gallinaceum and the 48,000/45,000 mol. wt antigens in P. falciparum (Kumar 1985, Vermeulen et al. 1985). In gel permeation chromatography all three polypeptides were found to be co-eluted. Cross-linking reagents were used to cross-link proteins on the intact cells and in the soluble extracts. The cross-linked product immunoprecipitated by the MoAbs showed an approximate mol. wt of 290,000 to 300,000, suggesting a stoichiometry of 1:1 between the high and low mol. wt polypeptides. Evidence was also obtained for the existence of a subpopulation of the 48,000/45,000 mol. wt polypeptides which is not complexed with the 230,000 mol. wt polypeptide. These data indicate that the 230,000 polypeptide is physically associated with the 48,000 and 45,000 polypeptides; the high and low molecular weight polypeptides appear to exist in a stable membrane bound complex.

Conditional absence of mitosis-specific antigens in a temperature-sensitive embryonic-arrest mutant of Caenorhabditis elegans.

A monoclonal antibody, specific to phosphoproteins in mitotic HeLa cells was found to crossreact with a similar set of proteins in embryos of the nematode, Caenorhabditis elegans. In C. elegans, as in mammalian cells, the highly conserved antigenic epitope is associated with a family of high molecular weight polypeptides. The antigenic reactivity of these multiple proteins also depends on their phosphorylation, since antibody binding is reduced after alkaline phosphatase treatment. The antigens are detected at the centrosomes, and in the nuclear region and surrounding cytoplasm of mitotic cells. The significance of these antigens is emphasized by their absence at restrictive temperature in embryos of the temperature-sensitive embryonic-arrest mutant, emb-29V. Furthermore, temperature shift-down experiments suggest that the emb-29 mutation defines a cell division cycle function that affects an essential activity required for progression into M phase.

Isolation and in Vitro Translation of Polysomes from Mature Rye Leaves

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.

Characterisation of aqueous extracts of seed proteins of Lupinus albus and Lupinus angustifolius

AbstractAqueous isolation of proteins from lupin seeds at different pH values has been examined. Protein isolates were characterised by gel filtration, electrophoresis and protein digestibility studies. The efficiency of extracting protein, expressed as solubilised nitrogen, from L. angustifolius and L. albus seeds can be manipulated by using aqueous media at pH <3 or >8. Although lupin proteins extracted at pH 2, 7 and 8.5 produced similar elution profiles on gel filtration analysis, lupin proteins extracted at pH 2 contained a low proportion of high molecular weight polypeptides, and those at alkaline pH, e.g. at 8.5, contained high molecular weight polypeptides similar to those extracted from the seeds directly. This situation was confirmed by SDS—PAGE analysis. It can be concluded that structural damage to the seed proteins occurs at pH 2 extraction, resulting in more unfolded conformation. This conclusion is consistent with our results from in vitro protein digestibility studies.

Electron spin resonance and spin-trapping studies of radiation damage in biologically significant molecules

Recent electron spin resonance (ESR) and spin trapping studies of γ-radiation induced radicals in protein and nucleic acid constituents in the polycrystalline state and in aqueous solutions are reviewed. When complex mixtures of spin-adducts are generated, the identification of radicals is facilitated by combining spin trapping with high pressure liquid chromatography, using ESR as an in-line detector. For high molecular weight polypeptides and polynucleotides, enzymatic hydrolysis of the spin trapped macromolecular radicals appears to be a promising approach. Attempts to spin trap radicals inside γ-irradiated cells and whole animals are discussed.

CDNA clone encoding a high molecular weight antigen of Babesia bovis

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate K A into bacteriophage λgt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the K A isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.

The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications

Chicken intestinal sucrase-isomaltase and maltase-glucoamylase have been isolated in their intact form by detergent solubilization and characterized as to their subunit composition and mode of anchoring in the brush-border membrane. Both are heterodimeric enzyme complexes composed of two subunits each of approximately 140 and 130 kDa. Contrary to the mammalian sucrase-isomaltase, chicken isomaltase was identified as the smaller of the two subunits. As was shown by hydrophobic labeling, only one of the two subunits in each heterodimer is anchored in the bilayer, the smaller 130 kDa isomaltase subunit of the sucrase-isomaltase complex, and the larger 140 kDa subunit of the maltase-glucoamylase complex. Both preparations contain a high-molecular weight polypeptide of approximately 250 kDa which in the case of sucrase-isomaltase could be identified by peptide mapping as a single-chain precursor not (yet) proteolytically processed to the final heterodimer. These first data on the mode of membrane anchoring of non-mammalian glycosidases indicate that they are synthesized, inserted into the membrane, and processed in ways similar to the mammalian enzymes. The fundamental unity between avian and mammalian sucrase-isomaltases suggests that the partial gene duplication of an ancestral isomaltase gene and the subsequent mutation of one of the active sites resulting in pro-sucrase-isomaltase has occurred prior to the separation of mammals from reptiles, i.e. more than 300 million years ago.