SOME CHARACTERISTICS OF THE SPERM ATTRACTANT FROM OEDOGONIUM CARDIACUM1

A bioassay for the sperm attractant from Oedogonium cardiacum was developed. The attractant was confirmed to be highly water soluble, destroyed by heating to 100 C and by dilute acid or alkali. It was fractionated by gel filtration with the attractant accompanied by yellow pigments. It has it molecular weight between 500 and about 1500, based on gel filtration, ultrafiltration, and dialysis. Freezing changes this to both larger and smaller molecules. It appears to have cationic groups. A paper chromatographic separation was found that divided the pigment into at least 3 major bands, one of which was closely associated with the attractant. An absorption spectrum of this pigment showed 1 peak in the visible spectrum at 417 mμ and 3 well-defined peaks in the ultraviolet region. The lack of a peak at 280 mμ indicates the attractant is not a polypeptide.     

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.     

Variability in the apparent molecular weight of inhibin

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in 1 M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in 0.1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.     

Activation of heparin cofactor II by heparin oligosaccharides

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.     

Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

Glycosaminoglycans from marine clam Meretrix meretrix (Linne.) are an anticoagulant

The extracted unfractionated glycosaminoglycans (GAGs) obtained from the marine clam Meretrix meretrix were fractionated by ion-exchange (Amberlite IRA-900 and 120) chromatography. The fractionated sample activity was determined through azure-A by metachromatic activity and agarose gel electrophoresis. The active fractionated sample molecular mass was determined through gradient polyacrylamide gel electrophoresis (PAGE). The structural characterization of low-molecular-weight GAGs was analyzed by Fourier transform infrared (FT-IR) and 1H-nuclear magnetic resonance (NMR) spectroscopy. The Activated partial thromboplastin time (APTT) of fractionated heparin-like glycosminoglycan is 72 IU/mg and it has a molecular mass of 15,000 Da. The disaccharide profiles, such as uronic acid 15.31%, hexosamine 24.61%, and sulfate content 11.7%, were also determined. The results of this study suggest that the GAGs from M. meretrix could be an alternative source of heparin.     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Prefractionation of protein samples for proteome analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of polyacrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 segments according to size, and each segment was ground in extraction buffer. The eluted proteins were separated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spectrometry. The molecular masses of the identified proteins in the > 48 kDa gel segment were distributed between 50 and 112 kDa, thus validating this prefractionation method. Identified HMW proteins with similar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides improved separation and detection of HMW proteins.     

5-lipoxygenase binds calcium

5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of leukotrienes and lipoxins and has therefore become an important target for pharmacological treatment of inflammatory disorders. Binding of calcium to 5LO was shown using several different approaches. Human recombinant enzyme was expressed in E. coli and purified. Association of Ca2+ to 5LO was demonstrated by a calcium-induced mobility shift in gel electrophoresis, by calcium overlay, by gel filtration in the presence of calcium, and by equilibrium dialysis. The two latter methods also showed that calcium binds reversibly to 5LO. Equilibrium dialysis gave a Kd close to 6 microM; the stoichiometry of maximum calcium binding seemed to average around two Ca2+ per 5LO. Similar results were obtained when 5LO was inactivated during equilibrium dialysis, indicating that the calcium binding site(s) is (are) different from the active site. By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO.     

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.     

Properties of membrane-inserted protein kinase C

Protein kinase C (PKC) interacted with phospholipid vesicles in a calcium-dependent manner and produced two forms of membrane-associated PKC: a reversibly bound form and a membrane-inserted form. The two forms of PKC were isolated and compared with respect to enzyme stability, cofactor requirements, and phorbol ester binding ability. Membrane-inserted PKC was stable for several weeks in the presence of calcium chelators and could be rechromatographed on gel filtration columns in the presence of EGTA without dissociation of the enzyme from the membrane. The activity of membrane-inserted PKC was not significantly influenced by Ca2+, phospholipids, and/or PDBu. Partial dissociation of this PKC from phospholipid was achieved with Triton X-100, followed by dialysis to remove the detergent. The resulting free PKC appeared indistinguishable from original free PKC with respect to its cofactor requirements for activation (Ca2+, phospholipid, and phorbol esters), molecular weight, and phorbol 12,13-dibutyrate (PDBu) binding. The binding of PDBu to free and membrane-inserted PKC was measured under equilibrium conditions using gel filtration techniques. At 2.0 nM PDBu, free PKC bound PDBu with nearly 1:1 stoichiometry in the presence of Ca2+ and phospholipid. No PDBu binding to the free enzyme was observed in the absence of Ca2+. In contrast, membrane-inserted PKC bound PDBu in the presence or the absence of Ca2+; calcium did enhance the affinity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.     

Thermodynamics of the binding of Ca2+ to porcine pancreatic phospholipase A2

The binding of Ca2+ to porcine pancreatic phospholipase A2 was studied by batch microcalorimetry. Enthalpies of binding at 25 degrees C were determined as a function of Ca2+ concentration in buffered solutions at pH 8.0 using both the Tris-HCl and Hepes-NaOH buffer systems. The calorimetric results indicate that protons are released on calcium binding and that in addition to the binding of the active-site calcium, there appears to be weak binding of a second Ca2+. Results from potentiometric titrations indicate that this proton release on binding Ca2+ arises from a change in pK of a histidine(s) functional group. The thermodynamic functions delta G0, delta H0 and delta S0 for calcium binding to phospholipase A2 have been determined. These results are compared with literature data for Ca2+ complex formation with some small molecules and also the protein troponin-C.     

Structure of bovine casein oligomers

We have characterized the size, molecular weight, and composition of the oligomeric particles produced by dialysis of bovine casein micelles against solutions lacking calcium ion. The particles were stabilized against further dissociation after dialysis by glutaraldehyde fixation. The progress of the dissociation was monitored by Biogel A-15 gel permeation chromatography, sucrose density gradient ultracentrifugation, inelastic laser light scattering, sedimentation velocity and equilibrium, and urea starch gel electrophoresis. The casein oligomers isolated after dialysis against either 0.5 m NaCl/SMUF A/azide or calcium-free SMUF/ azide have a hydrodynamic radius of about 5.5 nm and a molecular weight of about 95,000 corresponding to roughly four casein monomers (SMUF, simulated milk ultrafiltrate). The oligomers are highly hydrated and contain one-third of the calcium ion found in native micelles. During the course of dialysis, the micelles gradually break down into a broad distribution of intermediatesized particles and then into the oligomers described above.     

Size distribution of the closed circular deoxyribonucleic acid molecules of Bacillus megaterium: sedimentation velocity and electron microscope measurements

The circular deoxyribonucleic acid (DNA) of Bacillus megaterium was fractionated by sedimentation velocity on preparative zonal gradients. The fractions obtained were characterized by analytical sedimentation velocity analysis on neutral and alkaline sucrose gradients, and by contour length measurement by electron microscopy. Each fraction was found to contain circular molecules of one to three discrete sizes, in various combinations of covalently closed and open circular forms. Estimations of the molecular masses of these sizes gave values of 3.9, 6.2, 16.0, 31, and 60 million daltons for the major molecular species. Minor amounts of molecules of 7.6, 11.7, 47, 89, and 112 million daltons were observed in the electron microscope analyses. Length measurements of almost 600 molecules from the various fractions showed that all except six could be placed in distinct size classes. The distribution of molecular sizes in unfractionated circular DNA was shown to consist primarily of the two smallest size classes, although the relative proportions by weight of five of the classes were roughly equivalent.     

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM.

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 +/- 2800 in Tris - HCl buffer and 21 000 +/- 1000 in 6 M guanidine - HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10(-3) M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 - 10(6) M-1 for the apparent association constant and 4.4 - 10(-4) mol of Ca2+ bound per g of protein for high affinity bindings sites were estimated, on the basis of data from the equilibrium dialysis. The origin and possible biological role of this protein is discussed.     

Fractionation of spectrin by differential precipitation with calcium

The calcium-stimulated ATPase activity of spectrin can be substantially separated from the magnesium-stimulated activity by partial precipitation of spectrin with calcium. The precipitated Ca-ATPase fraction can be dissolved by dialysis against EDTA, and the resulting solution can be polymerized into characteristic spectrin fibrils. The supernatant contains Mg-ATPase and, either before or after dialysis, contains predominantly “torus” forms. The two fractions are essentially identical on gel electrophoresis, being composed primarily of the high molecular weight peptides with a constant level of minor bands. These results imply that the Ca-ATPase is associated firmly with the high molecular weight peptides.