High-resolution Gel Permeation Research Articles

Comprehensive non-target screening of biomagnifying organic contaminants in the Baltic Sea food web

High-resolution mass spectrometry (HRMS) based non-target screening (NTS) is a powerful approach for the simultaneous determination of multiple environmental contaminant classes in complex biota samples. In this study, trophic biomagnification factor (TMF) directed NTS was performed to find and (tentatively) identify known, emerging, and new chemical contaminants that are persistent and biomagnify in Baltic Sea biota. The investigated food web included seven species: one filter feeder (blue mussel, Mytilus edulis), two fish (eelpout, Zoarces viviparous; herring, Clupea harengus), two marine mammals (harbor porpoise, Phocoena phocoena; grey seal, Halichoerus grypus) and two birds (guillemot, Uria aalge; white-tailed sea eagle, Haliaeetus albicilla). The NTS procedure included extraction with organic solvent mixtures, two-step high-resolution gel permeation chromatography clean-up, Florisil® fractionation, gas chromatography (GC) HRMS analysis in electron ionization (EI) and electron capture negative ion chemical ionization (ECNI) modes, and NTS data processing. The latter was performed differently for the EI and ECNI data: the EI data were treated using a flexible and highly automated TMF-directed NTS workflow, whereas the ECNI data were treated with a simpler and less automated workflow that specifically screened for brominated compounds. The two workflows collectively revealed biomagnification (statistically significant TMF values) of >250 tentatively identified compounds, including legacy persistent organic pollutants (POPs), such as PCBs and PCB-related compounds, DDT and its metabolites, and organochlorine pesticides (OCPs), contaminants of emerging concern (CECs), and halogenated natural products (HNPs). Among the tentatively identified CECs, nine have not previously been reported in environmental biota samples. These included four polymer additives (used as antioxidants, rubber additives or plasticizers) and two cosmetic product additives (ethyl myristate and isopropyl palmitate). The CECs should be prioritized for future structure verification and quantification using reference standards.

Mechanistic study on laccase-mediated formation of Fe-OM associations in peatlands

Peatlands represent one of the largest terrestrial carbon pools. Phenol oxidase is widely considered to play a critical role in the decomposition of organic matter in peatlands, which is known as the “enzymatic latch”. However, our recent studies have indicated that the formation of iron-organic matter (Fe-OM) associations is more likely to occur in the presence of laccase, the largest class of phenol oxidases in soils. As the formation of Fe-OM associations is recognized as an important stabilization mechanism for soil organic matter, further clarification of the potential mechanisms contributes to a better understanding of the role of laccase in peatland carbon sequestration. In this study, a series of experiments using different phenolic compounds and peat-derived dissolved organic matter (DOM) were performed. The results from liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and gel permeation chromatography (GPC) analyses indicated that laccase catalyzed the polymerization of phenolics and peat-derived DOM by coupling of the C-O bond, the C–C bond and the C–C/C-O bonds. The polymerized products can combine more effectively with Fe to form Fe-OM associations. FTIR spectral analysis indicated that organic matter associated with Fe under laccase catalysis contained more oxygen-containing functional groups but fewer aromatic CC bonds. Although laccase catalysis can change the chemical structure of organic matter, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analyses showed minimal influence of laccase on the Fe chemical species in the Fe-OM associations. Thus, we propose that laccase mediated the formation of Fe-OM associations by polymerizing organic matter and by increasing the O/C ratio of organic matter. In addition, laccase can improve the sequestration of organic carbon by increasing the C/Fe molar ratio of the formed Fe-OM associations. This mechanism may provide further insight into the role of laccase in the carbon cycling in peatlands.

Study of the Chemical Structure of Dialdehyde Carboxymethyl Cellulose Produced by Periodate Oxidation under Different Conditions

13C nuclear magnetic resonance, paramagnetic resonance, and ultraviolet spectroscopy and high-resolution gel permeation chromatography were used to investigate the process of periodate oxidation of carboxymethyl cellulose and the structure and molecular weight characteristics of its oxidised forms produced under different conditions. It was shown that, in solution and in the solid state, aldehyde groups in the oxidised units exist in the form of cyclic hemiacetals. An examination was made of the existence of all the main cyclic intramolecular hemiacetal structures, and the most probable were proposed. The influence of the degree of periodate oxidation on the molecular weight characteristics and hydrolytic stability of dialdehyde carboxymethyl cellulose was studied.

Studies on the effect of concentration of formaldehyde on the synthesis of resole-type epoxidized phenolic resin from renewable resource material

Epoxidized cardanol/formaldehyde resins (ERCF) were synthesized by reacting resole type cardanol/formaldehyde resin and epichlorohydrin in basic medium at 120 °C. Resole-type phenolic resins were synthesized by reacting cardanol and formaldehyde (molar ratios 1:1.3, 1:1.5, 1:1.7, 1:1.9,and 1:2.1) in the presence of sodium hydroxide, as catalyst, at five different temperatures ranging between 60 and 80 °C with an interval of 5°C for a maximum period of 6 h. A set of samples were characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopic analysis. The molecular size distribution of resole phenolics was studied by high-resolution gel permeation chromatography. Differential scanning calorimetry technique was used to investigate the curing behavior of the synthesized epoxy resins. Single-step mass loss in dynamic thermo gravimetric traces for the ERCF was observed.

High Resolution Gel Permeation Chromatography Followed by GC–ECD for the Determination of Pesticide Residues in Soybeans

An efficient and validated approach for the determination of pesticide residues in soybeans using high-resolution gel permeation chromatography in combination with gas chromatography and electron capture detection is described. Gel permeation chromatography was used to remove interfering fatty components of soybeans before gas chromatographic analysis. The limit of quantification for the seven pesticides studied was between 9 and 46 μg kg−1. The method was applied to different soybean varieties and recoveries were determined to be between 93 and 118% with RSD values below 10%.

Identification of protein phosphatase 2C and confirmation of other protein phosphatases in the ocular lenses

The reversible phosphorylation of proteins plays essential roles in regulating various cellular events, and is regulated by the opposing actions of protein kinases and protein phosphatases. Protein kinases in the lens system have been well studied, but very little is known about lens protein phosphatases. Protein phosphatases can be divided several families, such as protein phosphatase types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C) and protein tyrosine phosphatases (PTP). In this study we evaluated what kinds of protein phosphatases are present in the lens by using various specific substrates and inhibitors. Samples were prepared from lenses of 17-day-old chick embryos, and fractionated by high-resolution gel permeation column chromatography, then the fractions were assayed for phosphatase activities. The results with 32P-labeled glycogen phosphorylase A, okadaic acid and inhibitor-1, which are a specific substrate and inhibitors of PP1 and/or PP2A, showed that PP1activities were present in the 500-, 115- and 45-kDa fractions of the lens protein. The 115-kDa fraction also contained PP2A activity. By using a phosphothreonine-containing peptide as a substrate, three peaks of phosphatase activities were found at around 115, 55 and 35 kDa. Based on their response to various phosphatase inhibitors and their metal dependency, the fractions of 115 and 35 kDa were concluded to contain PP2A, while the 55-kDa fraction contained PP2C. Immunoblot using specific antibodies against PP1, PP2A and PP2C confirmed that each fraction above contained corresponding protein phosphatases as proteins. When a phosphotyrosine-containing peptide substrate was examined at pH 7·4, we observed a major peak at 500 kDa, which was presumed to contain receptor-like PTP(s). On the other hand, at pH 5·5, we observed a peak of 18 kDa, which was confirmed to contain a low-molecular-weight PTP. These protein phosphatases have recently been suggested to be involved in stress response and apoptosis. Their physiological roles in the lens are of much interest.

Predominant phosphatase in the ocular lens regulated by physiological concentrations of magnesium and calcium

A magnesium-dependent phosphatase with a molecular weight of about 55 kDa was found in the lens of chicken embryo, mouse, rabbit and bovine. It appears to be unique to the lens and, when activated by magnesium, accounts for the majority of the phosphatase activity in the lens. Phosphatases in the lens were separated by using high-resolution gel permeation column chromatography, and phosphatase activity was determined with p-nitrophenyl phosphate ( pNPP) as a substrate. The 55-kDa phosphatase showed very low basal activity, but the activity was increased concentration-dependently by magnesium ion (Mg 2+), and at 1 mM Mg 2+, this enzyme accounted for over 50% of the total phosphatase activity in the lens. Calcium potently inhibited the magnesium-activated phosphatase activity in a dose-dependent (IC 50; about 50 μM), uncompetitive manner. The phosphatase activity was high in the acidic pH range, with an optimum pH value of 5.5. The characteristics of the lens Mg 2+-dependent phosphatase, such as substrate specificity and sensitivity to various phosphatase inhibitors, showed little similarity to those of any reported phosphatase. The Mg 2+ and Ca 2+ concentrations that were found to be effective in this study are similar to physiological concentrations in the lens; therefore it is likely that these ions physiologically regulate the 55-kDa phosphatase activity in the lens.

18 kDa Protein Tyrosine Phosphatase in the Ocular Lens

Protein tyrosyl phosphorylation and dephosphorylation play essential roles in regulating cellular events such as proliferation and differentiation, and their involvement in the lens development and transparency is also suggested. The level of tyrosine phosphorylation in a given protein is regulated by the opposing actions of protein-tyrosine kinases (Tyr kinases) and protein-tyrosine phosphatases (TyrPases). Recent studies have revealed that some Tyr kinases, such as platelet-derived growth factor receptor and fibroblast growth factor receptor, are present in the lens, however, little is known about TyrPases in the lens. In this study, we found a 18kDa protein tyrosine phosphatase (18kDa TyrPase) predominantly present in the ocular lens of various animals. We purified the phosphatase from the lens of chick embryo and characterized its activity.Phosphatase activity was determined in chick embryo, mouse, rabbit and bovine lenses usingp -nitrophenyl phosphate (p NPP) as substrate. All lenses examined dephosphorylated p NPP under acidic conditions, and a large portion of the activity resided in a low molecular weight protein, ca. 18kDa, following high-resolution gel permeation column chromatography. The brain and liver showed high dephosphorylation activities, but most of their activity was present in high molecular weight fractions, unlike that in the lens. The 18kDa phosphatase was purified from the lens of 17 day old chick embryos to near-homogeneity with two-step rapid chromatography. This phosphatase showed strict substrate specificity for phosphotyrosine and phosphotyrosyl peptides, suggesting that it was a kind of protein tyrosine phosphatases (TyrPases). Several known inhibitors of TyrPases, such as SH blockers, vanadate and phenylarsine oxide, strongly inhibited the enzyme activity. The molecular weight, substrate specificity, and responses to various inhibitors and activators coincide well with those reported for the low molecular weight protein tyrosine phosphatase (LMW-TyrPase), belonging to the TyrPase superfamily. These results suggest that the 18kDa phosphatase found in the lens is a LMW-TyrPase. The 18kDa TyrPase is the predominant phosphatase in the ocular lens. It may be involved in regulation of lens cell proliferation, differentiation and/or lens transparency.

Extraction and cleanup methods for analysis of phenolic and neutral organohalogens in plasma.

A method for the analysis of potential endocrine-disrupting compounds, such as phenolic halogenated compounds (e.g., chlorinated and brominated phenols) and hydroxylated PCBs, in blood plasma is presented. Neutral halogenated compounds, specifically brominated diphenyl ethers and PCBs, are also included in the evaluation. An efficient denaturation and extraction step is described, and three methods for lipid removal are evaluated. The latter includes a nondestructive method based on high-resolution gel permeation chromatography (HR-GPC), a newly developed silica gel/sulfuric acid column, and lipid removal by sulfuric acid treatment. Recoveries, based on gas chromatography with an electron capture detector (GC-ECD), were between 70 and 90% for most of the studied compounds. The recoveries of phenolic compounds were generally slightly lower than those of the neutral compounds. The sulfuric acid treatment and silica gel/sulfuric acid column gave the highest yields for acid stable compounds, although a few target compounds were lost during that treatment and all compounds were recovered with the HR-GPC method.

Adaptation and application of a multi-residue method for the determination of a range of pesticides, including phenoxy acid herbicides in vegetation, ased on high-resolution gel permeation chromatographic clean-up and gas chromatographic analysis with mass-selective detection

A multi-residue method to determine pesticides in vegetation samples is described. Blended samples were extracted with acetone and the pesticides partitioned into dichloromethane prior to being cleaned-up using high-resolution gel permeation chromatography. Investigations were made into the effects of altering the pH of the aqueous phase during partitioning. Rapid and efficient methylation of the phenoxy acids present was achieved using trimethylsilydiazomethane. The resultant solutions were then selectively and sensitively analysed by gas chromatography with mass-selective detection. The variation in results using matrix matched and non-matrix matched calibration standards was also explored. The method has been applied to grass samples spiked with pesticides from the following groups, phenoxy acid, organophosphorus, triazine, organochlorine, phenyl urea, chloroacetanilide and pyrethroid. Recoveries around 80–100% and reproducibilities of 2–4% ( n=4) were typically achieved at a spiking level ∼ 140 μg kg −1

Determination of phenoxy acid herbicides in vegetation, utilising high-resolution gel permeation chromatographic clean-up and methylation with trimethylsilydiazomethane prior to gas chromatographic analysis with mass-selective detection

A method for the determination of phenoxy acid herbicides in vegetation samples is described. Macerated samples are extracted with acetone and following filtration and acidification, the herbicides are partitioned into dichloromethane. The herbicides are then cleaned-up using high-resolution gel permeation chromatography before undergoing rapid and efficient methylation using trimethylsilyldiazomethane. The resultant methyl esters are then selectively and sensitively analyzed by gas chromatography with mass selective detection. Silanized glassware was used throughout. The method has been applied to grass samples spiked with four phenoxy acid herbicides; 2,4-D, dichlorprop, MCPA and mecoprop. Recoveries around 100% and reproducibilities of 5% ( n=4) were achieved at two spiking levels, ∼700 and ∼140 μg/kg.

Morphology and physical properties of poly(styrene-b-isobutylene-b-styrene) block copolymers

Abstract A series of linear and three-arm star poly(styrene-b-isobutylene-b-styrene) (PS-PIB-PS) block copolymers with varying block compositions was synthesized via living carbocationic polymerization using the initiation system 1,3-di(2-chloro-2-propyl)-5-tert-butylbenzene (or 1,3,5-tris-(2-chloro-2-propyl)benzene)/TiCl4/pyridine/2,6-di-tert-butylpyridine in a 60 40 (v/v) hexane/MeCl solvent mixture at −80°C. High resolution gel permeation chromatography showed that the compositions of the copolymers were complex, consisting of higher molecular weight coupled products, and products of lower molecular weight including, probably, homo-PS. Morphology and physical properties were characteristic of microphase-separated block copolymers, and were affected strongly by the PIB span molecular weight and volume fraction of PS. When the latter was in the range 0.20–0.42 vol%, the morphology was characterized by cylinders of PS in a continuous phase of PIB; however, at least two samples exhibited mixed morphologies in which regions of PS cylinders coexisted with regions of PS spheres and lamellae, respectively. Linear samples showed much better long-range morphological order than star-branched samples. The dynamic mechanical response of block copolymers consisted of separate PIB and PS relaxations whose relative intensities scaled well with copolymer composition. The low-temperature relaxation was broad, which is an inherent characteristic of PIB; the high-temperature PS relaxation was narrow, suggesting well defined phase separation and a sharp PIB-PS interface. Melt rheological studies of representative samples showed that microphase separation persisted up to 265°C. Tensile properties varied with PIB span molecular weight and PS content. An abrupt change occurred from 15–22 vol% PS, indicating a minimum PS content needed for strong network formation. Properties changed from elastomeric within the range 20–37 vol% PS, to ductile in the range 37–45 vol%, to brittle in the range 45–55 vol%. Samples with PS vol% ⩾ 35 yielded improved elastomeric properties as a result of annealing above the glass transition temperature of PS. Within the elastomeric range, the PIB span molecular weight was the dominant factor effecting elongation at break. A high tensile strength of 24 MPa was obtained, and most samples displayed strengths ⩾ 15 MPa.

An integrated strategy for structural characterization of the protein and carbohydrate components of monoclonal antibodies: application to anti-respiratory syncytial virus MAb.

The relatively rapid and extensive characterization of the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncytial virus (RSHZ19) is presented. The integrated strategy used a combination of mass spectrometric and conventional methodologies. Liquid chromatography/electrospray mass spectrometry was used for peptide mapping and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of selected peptides. Matrix-assisted laser desorption/ionization mass spectrometry provided the M(r) of the intact protein and was used to characterize endo- and exoglycosidase digests of isolated glycopeptides to identify the glycosylation-site peptide and define the structures of the carbohydrates at that site. These experiments verified 99.1% of the light- and 99.3% of the heavy-chain amino acid sequences. The N and C termini of both chains were confirmed, and the nature and extent of heterogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demonstrated by sequencing the N-terminally blocked peptide by tandem MS. Carbohydrate was found exclusively at Asn296 of the heavy chain. There was no evidence for a nonglycosylated form of the molecule or for the presence of O-linked carbohydrate. The qualitative distribution of glycoforms at this site was determined by MS of the isolated, tryptic glycopeptide and compared with results obtained by high-performance anion exchange chromatography and high-resolution gel permeation chromatography of oligosaccharides released by hydrazinolysis. The sequence and linkage of individual glycan species were determined using matrix-assisted laser desorption/ionization MS to monitor the results of a series of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated carbohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consistency of the MS methods with conventional methods for carbohydrate analysis.

Syntheses and characterization of UV‐curable polysiloxane‐based polyurethane prepolymers

AbstractUV‐curable polyurethane prepolymers were prepared from the following components: iso‐phorone diisocyanate, hydroxybutyl‐terminated polysiloxane, and 2‐hydroxyethyl meth‐acrylate with diethylene glycol or neopentyl glycol added as an optional chain extender. These prepolymers, with hard segments of different structures, were characterized by high resolution NMR, FTIR, and gel permeation chromatography. Model oligomers were prepared to confirm the structures of the prepolymers. The structure–property relationship of cured prepolymers were determined. © 1995 John Wiley & Sons, Inc.

Structural analysis of tamarind seed xyloglucan oligosaccharides using β-galactosidase digestion and spectroscopic methods

The borohydride-reduced forms (oligoglycosyl alditols) of two isomeric octasaccharides (Glc 4Xyl 3Gal) that are released from xyloglucans of various plant species upon treatment with a fungal endo-(1 → 4)-β-glucanase were isolated and structurally characterized. A mixture of oligosaccharides that is released from tamarind seed xyloglucan by the endo-(1 → 4)-β-glucanase was digested with a commercially available β-galactosidase ( Aspergillus niger). The β-galactosidase selectively hydrolyzed the galactosyl residue of one of the two isomeric octasaccharides present in the mixture. A homogeneous preparation of the β-galactosidase-resistant octasaccharide was prepared by high-resolution gel-permeation chromatography of the enzyme-digestion products. Spectroscopic characterization of the oligoglycosyl alditol prepared by reduction of this octasaccharide confirmed the previously proposed structure that had been based on analysis of the mixture of isomeric octasaccharides. The availability of large amounts of the pure, reduced octasaccharide and of a pure, reduced pentasaccharide (Glc 3Xyl 2) made it possible to completely assign their 1H and 13C NMR spectra. In addition, the borohydride-reduced form of the β- d-galactosidase-susceptible octasaccharide isomer was purified by high pH anion-exchange chromatography of the endo-(1 → 4)-β-glucanase-released octasaccharides from rapeseed xyloglucan (no β-galactosidase treatment), and its 1H and 13C NMR spectra were assigned. Additional correlations between specific structural features of xyloglucan oligoglycosyl alditols and the positions of specific resonances in their NMR spectra were deduced and added to the extensive list that we have compiled. The effects of recording the NMR spectra of the xyloglucan oligoglycosyl alditols in the presence of borate salts, which could lead to incorrect structural assignments, are also described.

Characterisation of oligosaccharides from Drosophila melanogaster glycoproteins

An analysis of the released oligosaccharides from a membrane glycoprotein preparation of third instar larvae (3rdIL), and purified larval serum protein 2 (LSP2) from Drosophila melanogaster was performed. Sequential exoglycosidase digestion in combination with high-resolution gel permeation chromatography and partial acetolysis indicated the presence of two series of oligomannosides; one of these series was unusual and characterized by the presence of a core α1–6 linked fucose, the other was a typical mammalian oligomannose series containing the following isomers −D 1, −D 2, −D 12, −D 123 and −CD 123 as well as the unprocessed Man 9GlcNAc 2 structure. Conventional oligomannose could only be detected in the LSP2 sample. This study opens the way to use powerful molecular and classical genetic techniques to analyse the control and functional significance of glycosylation in higher organisms.

Purification and partial characterization of stonustoxin (lethal factor) from Synanceja horrida venom

1. 1. The lethal factor of the stonefish ( Synanceja horrida) venom, designated as the stonustoxin, was purified to homegeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated α and β) with mol. wts of 71,000 and 79,000, respectively. 4. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the β subunit were also determined. 5. 5. Purified stonustoxin had an LD 50 of 0.017 μg/g which is 22-fold more potent than that of the crude venom. 6. 6. The toxin exhibited potent haemolytic activity in vitro and endema-inducing activity with a minimum edema does (MED) of 0.15 μg in mouse paw. The edema effect was not antagonized by diphenhydramine.

The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered

Desmin protofilaments and the proteolytically derived alpha-helical rod domain have been characterized by high-resolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 +/- 5 nm and a tetramer species (two coiled coils) with a length of 65 +/- 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The data on the hydrodynamic analysis of the rod tetramer of desmin in solution are in full agreement with the structural information recently deduced from paracrystals of the rod of glial fibrillary acid protein [Stewart, M., Quinlan, R.A. & Moir, R.D. (1989) J. Cell Biol. 109, 225-234]. Our results explain the inhomogeneity of molecules encountered in previous electron microscopical analyses.

Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.

Isolation and partial characterization of an 80,000-dalton protein kinase from the microvessels of the porcine brain.

A novel serine/threonine-specific protein kinase was isolated from the microvessels of porcine brains. The molecular mass of the protein is 80,000 daltons, as judged by gel electrophoresis under denaturing conditions, or 122,000 daltons, on high-resolution gel permeation chromatography in the native state. The activity of this enzyme is stimulated by various histones or polyamines, like spermine or spermidine, but not by any of the common second messengers. The amino-terminal sequence data show no homologies to any of the published kinases, but rather to a heat-shock protein of unknown function.