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J F Hainfeld

Publications and source records attributed to J F Hainfeld.

At least 55 records · Page 3Linked to original sources
PubMed1990

Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.

Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the same core features as fVIII molecules. Molecular species corresponding to heterodimers (mass, 128 +/- 13 kD) and unassociated subunit chains (40-100 kD) were also observed in fVIIIa preparations, suggesting that heterotrimers have an appreciable tendency to dissociate, a phenomenon that could explain the decay of fVIIIa activity after thrombin activation of fVIII.

Animals↗
PubMed1990

Eleven tungsten atom cluster labels: high-resolution, site-specific probes for electron microscopy.

Two derivatives of a tungstate cluster containing 11 tungsten atoms (W11PO39SiR4-) have been synthesized which enable them to be covalently attached to biomolecules at specific sites. The tungstate cluster is 1.0 nm in diameter, electron dense, and visible in the electron microscope. One derivative is a W11-sulfonyl chloride, reactive with amines and sulfhydryls. The second compound is a W11-thiosulfonate which can be used to label sulfhydryl groups. These new labels are beam resistant and provide significantly higher resolution then most other electron microscopy (EM) markers. Labeling of the protein albumin is described as an example.

Cations↗
PubMed1990

A high-resolution tungstate membrane label.

A new class of membrane labels was synthesized which contain a tungstate cluster (having 11 tungsten atoms) and an aliphatic organo-tin moiety with various chain lengths (C4, C8, C12, C18, C22). These molecules were found to insert into synthetic phospholipid vesicles and biological membranes (human red blood cell membranes). The tungstate clusters can be individually visualized in the high resolution STEM or seen en mass in thin-sectioned labeled membranes in the CTEM. These new labels should provide a means for direct high-resolution imaging of lipid-phase systems.

Cell Membrane↗
PubMed1989

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit.

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Buffers↗
PubMed1989

Branched-chain alpha-keto acid dehydrogenase complex from bovine kidney: radial distribution of mass determined from dark-field electron micrographs.

Scanning transmission electron microscopy (STEM) was used to determine the radial distribution of mass within the bovine kidney branched-chain alpha-keto acid dehydrogenase complex (E1-E2) and its core enzyme, dihydrolipoamide acyltransferase (E2). The particle mass of E2 measured by STEM is (1.19 +/- 0.02) x 10(6). Assuming 24 subunits per E2 core, this value corresponds to a subunit molecular weight of (4.96 +/- 0.08) x 10(4), which agrees well with the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 5.2 x 10(4) (Pettit et al., 1978) and that deduced from the gene sequence, 46,518 (Griffin et al., 1988). Thus, the STEM data reaffirms the 24-subunit model for this E2. Previous studies indicated that the E2 subunits contain an extended, outer lipoyl-bearing domain connected by a trypsin-sensitive segment to a compact, inner catalytic domain. The assemblage of 24 inner domains comprises a cubelike inner core. The quantity and spatial distribution of mass determined from STEM images for the E2 inner core are consistent with this model. The lipoyl-bearing domains are shown to occupy a zone defined by radii of 80-130 A over which the lipoyl moiety may range. This zone overlaps the positions of the 24 branched-chain alpha-keto acid dehydrogenase (E1) molecules, which apparently are located on the of the cubelike inner core.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1988

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy.

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.

Escherichia coli↗
PubMed1988

Titin: quantitative mass measurements by scanning transmission electron microscopy and structural implications for the sarcomere matrix of skeletal muscle.

Scanning transmission electron microscopy has been used to investigate mass and linear mass density of native titin-2, a large soluble fragment of intact titin, from rabbit skeletal muscle. Dark field images of unstained, freeze-dried titin-2 appeared as either compact globules or looser and larger balls of string. Direct mass measurements indicated that the compact forms have an average mass of 2.40 +/- 0.50 x 10(6) Da. The mass to length ratio, determined from well-spread portions of titin strands (3-5 nm wide) from the ball of string forms, averaged 2.7 +/- 0.9 kDa/nm. Thus a single native intact titin molecule has a calculated contour length of well above approximately 1 micron, sufficient to span unidirectionally between the Z line and M line region in a resting-length sarcomere.

Animals↗
PubMed1988

Gold cluster-labelled antibodies.

The Fab' fragments of antibodies can be combined with eleven gold-atom clusters to produce the smallest gold-conjugated antibody probes yet developed.

Animals↗
PubMed1988

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7.

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Amino Acid Sequence↗
PubMed1988

Membrane contents of distinct subpopulations of coated vesicles determined by scanning transmission electron microscopy.

In order to investigate the heterogeneity of clathrin-coated vesicles purified from rat liver, and to quantitate rigorously their membrane contents, we have analyzed scanning transmission electron micrographs of unstained coated vesicles before and after extraction with the non-ionic detergent Triton X-100, as well as of vesicles whose coats had been removed by dialysis against 10 mM or 100 mM Tris (pH 8.2). Their respective distributions of particle masses were thus determined and compared, in light of complementary biochemical quantitations of lipid and protein. Smaller coated particles, 25-45 MDa in mass and 60-80 nm in diameter, lose no mass when extracted with Triton, and disappear when their coats are dissociated. We conclude that they do not contain membrane vesicles, although they have dense, presumably proteinaceous, cores. They may represent particles generated during tissue homogenization or, possibly, a storage form of clathrin. The remaining 70% contain bona fide vesicles: these particles are 75-150 nm in diameter, and their average mass is about 80 MDa, of which 48 MDa is contributed by coat proteins, 10-12 MDa by phospholipid and cholesterol, and 20-22 MDa by vesicle-associated proteins. Their vesicles are of two types: smaller, denser, vesicles that contain substantial amounts of internalized material, and larger, less dense, vesicles that do not. The distinction between them may, in view of other findings, reflect a difference between coated vesicles derived respectively from the Golgi and the plasma membrane.

Animals↗
PubMed1987

A small gold-conjugated antibody label: improved resolution for electron microscopy.

A general method has been developed to make the smallest gold-conjugated antibody label yet developed for electron microscopy. It should have wide application in domainal mapping of single molecules or in pinpointing specific molecules, sites, or sequences in supramolecular complexes. It permits electron microscopic visualization of single antigen-binding antibody fragments (Fab') by covalently linking an 11-atom gold cluster to a specific residue on each Fab' such that the antigenic specificity and capacity are preserved. The distance of the gold cluster from the antigen is a maximum of 5.0 nanometers when the undecagold-Fab' probe is used as an immunolabel.

Ferritins↗
PubMed1987

Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli.

The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase. Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric). The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens. Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule. Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.

Escherichia coli↗
PubMed1987

Visibility and stability of a 12-tungsten atom complex in the scanning transmission electron microscope.

A complex consisting of 12 tungsten atoms has been studied in terms of signal-to-noise (S/N) and dose response in the scanning transmission electron microscope (STEM), to evaluate its suitability for use as a approximately 1 nm resolution biological label. Molecular weight of the complex was measured as a function of radius of integration, and results were in agreement with the calculated formula weight. S/N was highest at the lowest radius of integration (0.25 nm), and decreased monotonically with increasing radius. The complex was clearly visible at a dose of 4 X 10(3) e/nm2, and exhibited negligible mass loss (approximately 8%) after an accumulated dose of 1.28 X 10(5) e/nm2. Beam-induced motion was small, 0.46 nm rms after 4 X 10(4) e/nm2. Some intensity fluctuations were observed between successive scans of the same clusters, for which a diffraction-based explanation is advanced. Upon suitable functionalization, the tungsten complex is expected to complement the undecagold cluster already in use for site-specific labeling.

Chemical Phenomena↗
PubMed1987

The gene-specific initiation factor USF (upstream stimulatory factor) bound at the adenovirus type 2 major late promoter: mass and three-dimensional structure.

The gene-specific transcription initiation factor USF (upstream stimulatory factor) binds at a palindromic sequence that extends from -52 to -63 relative to the start site of the adenovirus type 2 major late promoter; USF enhances in vitro transcription 10- to 20-fold. By analysis of digital micrographs from the Brookhaven scanning transmission electron microscope, we have identified a sample of 29 proteins (mass, 55 +/- 5 kDa) specifically bound at the palindrome. The individual protein digital images show extensive homology, which permits modeling a three-dimensional structure at a relatively low resolution, which is nonetheless significant for the study of protein-protein interactions in initiation. Non-sequence-specific competitor DNA at high mass excess can be used in reactions for microscopy, enabling characterization of specific binding for proteins present at 1% of total protein or less.

Adenoviridae↗
PubMed1986

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy.

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).

Animals↗