Changes In Molecular Size Research Articles

The functional site of placental anticoagulant protein: Essential histidine residue of placental anticoagulant protein

Placental anticoagulant protein (PAP) rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH 7.9 and 8 °C. Photooxidized PAP failed to bind the phospholipid vesicle. It seemed unlikely that the protein underwent a change in molecular size during the photooxidation on the basis of its behavior in electrophoresis and gel filtration. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. The peptide, SHLRKV, was included in the functional site of PAP and still showed an anticoagulant activity. On the other hand, the peptide which substituted histidine by alanine, SALRKV, no longer showed the activity. It was shown that the histidine residue is involved in Ca 2+ or the phospholipid binding site of the protein.

Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals

Senescence of carnation (Dianthus caryophyllus L. ev. White Sim) petals coincided with a decrease on a per flower basis in the yield of cell wall and ethanol‐insoluble solids. The decrease in cell wall yield per flower was due largely to a loss of neutral sugars, primarily galactose (45%) and arabinose (23%). On a per flower basis, water‐and chelator‐soluble pectins increased throughout development, comprising in senescent petals 18 and 58%, respectively, of total pectin. Alkali‐soluble pectins ranged from 35 to 45% of the total pectin and decreased during senescence. Gel chromatography of chelator‐ and alkali‐soluble pectins revealed no change in molecular size and polygalacturonase activity was not detected. Large‐molecular‐size hemicelluloses decreased during development, an observation reminiscent of the changes affecting hemicelluloses during the ripening of a number of fruit types. Compositional analysis of the large hemicellulosic polymers revealed a decrease in xylose and galactose content.

On the photolysis of poly-4-chloromethylstyrene and poly-4-chloromethylstyrene-co-styrene in solution

Poly-4-chloromethylstyrene (PCMSt) and copolymers (CP-CMSt-St) of 4-chloromethylstyrene (CMSt) and styrene (St) were irradiated in dilute tetrahydrofuran solution with UV light. Continuous irradiation of PCMSt at λ inc = 254 nm resulted in intermolecular crosslinking and formation of HCl. Flash photolysis at λ inc = 266 nm gave evidence of benzyl type radicals by the characteristic optical absorption spectrum ( φ(benzyl) = 0·3 ± 0·1). From light scattering and optical absorption measurements it was inferred that, apart from benzyl radicals, other unidentified radicals also contributed to crosslinking. In the presence of O 2, PCMSt predominantly underwent mainchain scission. Crosslinking was observed with CP-CMSt-St containing 28 mol% CMSt but no change in molecular size was detectable with a copolymer containing only 6 mol% CMSt. Excimers of styrene repeating units were very effectively quenched by CMSt repeating units. This reaction resulted in the formation of additional benzyl type radicals. Exciton migration along the chain in segments consisting of styrene units is assumed to promote quenching.

CHARACTERIZATION OF THE ACTION OF BACILLUS SUBTILUS ALPHA-AMYLASE ON SWEET POTATO STARCH, AMYLOSE AND AMYLOPECTIN

The action pattern of Bacillus subtilis alpha-amylase on Jewel variety sweet potato starch, amylose and amylopectin was monitored using high performance size exclusion chromatography. A wide range of products, from high molecular weight polysaccharides to specific saccharides of low molecular weights, were observed. The hydrolysis pattern for amylose in starch was concealed by hydrolytic fragments formed from amylolysis of the greater quantity of amylopectin. Amylopectin was degraded to a more specific mixture of polysaccharide fragments. Changes in molecular size of hydrolytic fragments formed during amylolysis supported the cluster model previously proposed for amylopectin. These results also suggest that the action of alpha-amylase on sweet potato starch is generally a nonrandom process, primarily resulting from enzymatic action on amylopectin. To support this hypothesis, a more random pattern of hydrolysis was observed from acid treated starch.

Synthesis and intracellular processing of aminooligopeptidase by human intestine

Aminooligopeptidase is an intrinsic glycoprotein of the brush border membrane important for hydrolysis of the oligopeptide products of intraluminal protein digestion. To study its synthesis and intracellular processing, we performed pulse-chase experiments using [35S]methionine to label proteins of cultured human intestinal expiants obtained by endoscopic biopsy. Aminooligopeptidase was isolated by immune precipitation with a monoclonal antibody and its molecular size was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A precursor of relative molecular weight (Mr) 127,000 appeared within 10 min of chase and appeared to begin conversion to an Mr 150,000 form (the size of brush border membrane aminooligopeptidase) within 60 min. To determine if the change in molecular size was the consequence of alterations in glycosylation, we studied the susceptibility of the two forms to endo-β-N-acetylglucosaminidase H, which cleaves immature high-mannose N-linked carbohydrate chains, and to peptide: N4-(N-acetyl-β-glucosaminyl)asparagine amidase, which cleaves both the highmannose and complex N-linked carbohydrate chains. Only the early Mr 127,000 aminooligopeptidase was sensitive to endo-β-N-acetylglucosaminidase H, suggesting that the larger form results from trimming of high-mannose cores and adding terminal sugars in the Golgi complex. Both forms were sensitive to peptide:N4-(N-acetyl-β-glucosaminyl)asparagine amidase, generating an Mr 114,000 species. The kinetics of the synthesis and processing of aminooligopeptidase and sucrase-isomaltase were compared by immunoprecipitation of both proteins from the same tissue after separating the microvillous membrane from the remainder of the cellular membranes. Labeled aminooligopeptidase was present intracellularly in its mature form within 60 min and was detected exclusively in the brush border membrane by 90 min. Most of the labeled sucrase-isomaltase pool had not yet undergone complex glycosylation during the same period. These data demonstrate that although human intestinal aminooligopeptidase undergoes N-linked glycosylation like sucrase-isomaltase, the synthesis of aminooligopeptidase differs from that of sucrase-isomaltase in respect to the absence of a highmolecular-weight precursor and more rapid pre-Golgi processing.

Simultaneous observation of positive and negative nuclear overhauser effects in oligopeptides due to segmental motion

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides Boc-Cys-(Val) n-Trp-OMe (n=1–3) in dimethylsulfoxide, Boc-Cys-(Val) n-Trp-OMe have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C α H and C βH 2 resonances in the peptides where n=1 and 2. Negative NOEs are observed between backbone NH and C α H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

Change in Molecular Size of Mouse Mammary Gland Lipase with Parturition

Change in Molecular Size of Mouse Mammary Gland Lipase with Parturition

Activation affects access to the platelet receptor for adhesive glycoproteins.

Blood platelets have a receptor for macromolecular adhesive glycoproteins, located on a heteroduplex membrane glycoprotein complex (GPIIb/IIIa) that only becomes "exposed" when platelets are activated. Binding of the adhesive glycoproteins, in particular fibrinogen, to the receptor is required for platelet aggregation, which in turn is required to arrest bleeding. A murine monoclonal antibody whose rate of binding to the receptor is affected by platelet activation was both cross-linked and fragmented to assess the effects of changes in molecular size on its rate of binding to unactivated and activated platelets. The results indicate that small molecules can bind more rapidly to the receptors on unactivated platelets than can large molecules and that activation involves a conformational and/or microenvironmental change that permits the large molecules to bind more rapidly.

Scanning molecular sieve chromatography of interacting protein systems: Nonlinear least-squares analysis of small zone experiments

A simple routine for nonlinear least-squares analysis is applied to small zone scanning data, where calibration of the gel column requires the use of fully characterized markers of known molecular size. Application of nonlinear least-squares analysis eliminates the difficulty encountered because of scarcity of calibrating markers for gels whose porosities span certain size ranges by providing a sensitive measure of changes in molecular size of the interacting protein system, in order to determine the interaction parameters, size heterogeneity, and the centroid position for time difference chromatographic experiments.

Photo-Induced Wettability Change in Films Containing Spiropyran Microcrystals

Photo-induced wettability changes in polymer films containing spiropyrans with long alkyl chains are investigated as a function of alkyl chain length and spiropyran content. It was found that spiropyrans with longer alkyl chains easily crystallize in polymer films, leading to a greater change in wettability. It was also found that the size of the formed microcrystals plays an important role in exhibiting photo-induced wettability changes. The wettability change disappeared after repeated photo-irradiation because stress caused by the change in molecular size pulverizes the microcrystals into smaller particles which have no influence on wettability change.

Degradation of Wheat Starch in a Single‐Screw Extruder: Mechano‐Kinetic Breakdown of Cooked Starch

ABSTRACTWheat starch was processed in a 19 mm diameter split‐barrel laboratory extruder under varying conditions of barrel temperature (79–121oC), screw speed (50, 100 rpm) and moisture content (25, 30%). Final product and samples removed from along the barrel length were examined for changes in molecular size by dilute solution viscometry. Final products were also examined for paste viscosity and estimated degree of cook. A mathematical model was developed which relates the residence time of the starch in the barrel, the nominal shear stress acting on the starch and the degree of cook of the product to the extent of molecular degradation of the starch. Solution viscometry was found to be more rapid than gel permeation methods previously used to determine the extent of molecular changes.

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

Large photo-induced thickness change in evaporated spiropyran films

Evaporated spiropyran films exhibit a large photo-induced thickness change, which is caused by the change in molecular size associated with ring-opening.

Changes in Molecular Size and Chemical Properties of Gelatin Caused by the Reaction with Oxidizing Methyl Linoleate

Changes in Molecular Size and Chemical Properties of Gelatin Caused by the Reaction with Oxidizing Methyl Linoleate

Hormonal regulation of deoxyribonucleic acid polymerase beta activity in rat testis.

The content of DNA polymerase beta in whole rat testes drastically decreased upon hypophysectomy: at 1 and 2 weeks, to 12.5% and 5% of the control testes of sham-operated rats, respectively. DNA polymerase beta activity per cell (per milligram DNA) also decreased to 14% of the control within 2 weeks after hypophysectomy. The DNA polymerase alpha in the testis also decreased, but the DNA polymerase gamma was relatively resistant to hypophysectomy. The testicular DNA polymerase beta from hypophysectomized rats sedimented at 4.5 S, whereas that from sham-operated controls sedimented at 3.3 S. The reduced level of testicular DNA polymerase beta and the change in molecular size induced by hypophysectomy were largely reversed by daily injections of LH and FSH. The reduced activity was also fully reversed by the injection of testosterone. These results suggest that the level of DNA polymerase beta in the rat testis depends largely on the level of testosterone produced by Leydig cells in the testis itself, but is also regulated by pituitary gonadotropins.

Changes in molecular size and chemical properties of gelatin caused by the reaction with oxidizing methyl linoleate.

Gelatin, soy protein, lysozyme, succinyl-casein and succinyl-egg albumin were allowed to react with methyl linoleate (ML) at a relative humidity (RH) of 0% at 50°C for 7 days (protein: ML = 1:1). Gel filtration indicated that only gelatin was extensively fragmented. The gelatin was then incubated with ML under various conditions, and changes in the molecular sizes, the gel forming abilities and the chemical characteristics were investigated. The fragmentation of gelatin was increased by decreasing the RH and with the increase in the ratio of ML to protein. The melting point of gel in heating and cooling gelatin was decreased by increasing the fragmentation. The contents of amide and carbonyl groups increased and that of amino group decreased as the reaction progressed at RH 0%, but no change in C-terminal amino acids was observed. Following the reaction at RH 0%, many kinds of amino acid residues of gelatin were damaged, although in our previous paper [Matoba et al.,Agric. Biol. Chem. , 46, 979 (1982)] su...

Characterization of the glucocorticoid receptor translocated to the nucleus in intact AtT-20 mouse pituitary tumor cells at low temperatures.

Although low temperatures retard the nuclear translocation of glucocorticoid-receptor complexes, they do not completely inhibit it. To gain insight into the mechanism of nuclear translocation, we determined the Stokes radius and surface charge density of the receptors bound to the nucleus at 0 C and compared them with the values obtained from standard preparations of activated and unactivated glucocorticoid receptors. Specifically, intact, viable AtT-20 cells were incubated at 0 C for 3 h with 10 nM tritiated triamcinolone acetonide and the nuclear receptor extracted with 0.3 M KCl/20 mM sodium molybdate. These nuclear receptors were found to elute from an Agarose A 1.5 m column with a Stokes radius identical to that of an "activated" receptor (6.4 nm) but eluted from a DEAE column at the same position as an "unactivated" species (0.17 M KCl). From these results we hypothesize that the change in receptor structure that leads to nuclear binding is a multistep process, and although the change in molecular size may be a prerequisite for nuclear binding, the change in surface charge is not.

Critical aspects of immune complex assays employing polyethylene glycol

Treatment of artificial immune complexes (ICs) with 2.5% polyethylene glycol (PEG) — conditions under which C1q-binding activity is routinely measured in the fluid phase — produced marked changes in molecular size as determined by Sepharose 6B chromatography. The effect of PEG on the C1q-binding capacity of ICs, was therefore investigated using a solid phase (SP) system. PEG enhanced the binding of aggregated human gammaglobulin (AHG) and artificial ICs to SP-C1q and, in reverse experiments, also increased the binding of C1q to SP-AHG. The degree of enhancement varied according to the Ag: Ab ratio employed; the binding of ICs formed in moderate Ab excess was only modestly enhanced but that of complexes formed at slight Ab excess, equivalence and Ag excess was markedly elevated. The profile of PEG-induced enhancement of binding paralleled that of similar ICs in the C1q fluid phase system, suggesting that C1q binding in the latter may be influenced by PEG. However, the C1q-binding activity of in vivo-formed ICs seemed to be relatively unaffected by PEG since enhanced binding was comparable in control and pathological sera. The results indicate that PEG causes cross-linking and aggregation of ICs (and possibly other serum proteins) which may alter their biological activity and hence influence the results of IC assays that employ this agent.

The active site of carboxypeptidase Cu. II. Photooxidation of carboxypeptidases Cua and Cub.

Carboxypeptidases CUa and CUb were both inactivated by photooxidation in the presence of methylene blue at pH 5.5 and 8°C. The inactivation was partially prevented by a competitive inhibitor, 3-phenylpropionic acid, of the enzymes. It seemed unlikely that the enzymes underwent a change in molecular size during the photooxidation on the basis of their behavior in electrophoresis and gel filtration. When the photooxidation was carried out at various pH value ranging from 4.5 to 7.5, the rate of inactivation was found to be pH-dependent and the pH profiles conformed to theoretical titration curves with an apparent pKa value of 6.5, suggesting that an imidazole group of a histidine residue is essential for the enzymatic activity. The photooxidized enzymes had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. It was shown that one histidine residue is involved in each active site of the enzymes.

Immunology and immunopathology of trimellitic anhydride pulmonary reactions

Inhaled trimellitic anhydride (TMA) reacts with airway proteins to produce trimellityl (TM) proteins. The TM-proteins result in both systemic and local immune responses, of which various proteins present in the airway can be used for markers. Thus TM-human serum albumin (HSA), TM-IgG, and TM-IgA can be used as hapten-protein complexes for immunologic studies in sera of humans exposed to TMA by inhalation. Various immunologic assays have been established to measure antibodies against TM-proteins and have various purposes. With TM-HSA as a model antigen, total serum antibody may be measured by the ammonium sulfate technique of coprecipitation of TM- 125I HSA. By solid-phase radioimmunoassays, IgE, IgG, IgA, and IgM antibodies can be measured. Lymphocyte reactivity can be measured by 3H thymidine uptake of TM-protein-stimulated lymphocytes. Biologic effects of IgE antibody can be measured by allergy skin tests and leukocyte histamine release with TM-proteins such as TM-HSA. The reaction of TMA with proteins results in alteration of those proteins that include changes in charge and physical conformation, the latter resulting in an apparent change in molecular size. These changes may relate to the observations that human antibody is not merely directed against the hapten in the hapten-human protein complex but also against new antigenic determinants formed by the TM-protein complex. Correlations have been made with certain human immunologic responses and lung disease after TMA inhalation, as follows: IgE antibody against TM-proteins correlates with TMA-induced rhinitis, conjunctivitis, and asthma; high levels of total antibody, IgG, and IgA antibody appear to correlate with the late respiratory systemic syndrome, probably a variant of hypersensitivity pneumonitis; workers exposed to TMA fumes (rather than TMA powder) have the highest levels of antibody, and this may correlate with occurrence of the hemorrhagic pneumonitis seen in this group of workers; patients with no symptoms or mild irritative symptoms have the lowest or no antibody levels. The immunopathogenetic relationships may be better understood with the further development of animal models of TMA lung disease now available.