Biomedical subjects

J F Hainfeld

Publications and source records attributed to J F Hainfeld.

At least 19 recordsLinked to original sources

PubMed1995

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

Binding Sites↗

PubMed1994

Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster labels and scanning transmission electron microscopy.

This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E2E3 subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively. The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3. Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex. Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.

Escherichia coli↗

PubMed1994

An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.

An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

Amino Acid Sequence↗

PubMed1992

Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy.

A method is described to deliver 235U to tumors; the isotope would then be fissioned by incident neutrons, producing localized lethal radiation sufficient for therapy. Apoferritin was loaded with an average of approximately 800 238U atoms per molecule. Stability of the loaded apoferritin in solution was improved, so that only 8% loss of uranium occurred after 8 days at pH 7. Fab' antibody fragments were covalently attached to the uranium-loaded apoferritin, and the immunoreactivity of the conjugate was 92% of that for antibody alone. Such bio-uranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy.

Apoferritins↗

PubMed1992

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.

Animals↗

PubMed1991

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent significant conformational changes that were governed by the cooperative interactions of the ribosomal proteins. The sequential association of the first 12 proteins with the 16S rRNA resulted in the formation of core particles containing up to three mass centers at distinct stages of the assembly process. These globular mass centers may correspond to the three major domains (5', central, and 3') of the 16S rRNA. Through the subsequent interactions of the late assembly proteins with the 16S rRNA, two of the three domains merge, yielding the basic structural traits of the native 30S subunit. The fine morphological features of the native 30S subunit became distinctly resolved only after the addition of the full complement of proteins. The fully reconstituted 30S subunits are active in polyphenylalanine synthesis assays. Visualization of the assembly mechanism of the E. coli 30S ribosomal subunit revealed domain-specific folding of the 16S rRNA through the formation of distinct intermediate core particles hitherto not observed.

Bacterial Proteins↗

PubMed1991

Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster.

Scanning transmission electron microscopic images of transfer RNAs reveal the molecular dimensions and compact morphology of these small macromolecules in unprecedented detail. Selective labeling of a sulfhydryl group on 2-thiocytidine enzymatically inserted at position 75 at the 3' end of yeast tRNA(Phe) with an undecagold cluster permits identification of this specific tRNA site by dark field STEM. Imaging of a single nucleotide at a defined location on the tRNA molecule should make it possible to localize in situ tRNAs at the A, P, and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. In addition, this approach may be used for the highly specific topographical mapping of other RNAs and/or biological macromolecular complexes.

Gold↗

PubMed1991

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.

Gene Products, env↗

PubMed1990

Structural model of factors V and Va based on scanning transmission electron microscope images and mass analysis.

Coagulation factor V (fV) is a single-chain glycoprotein (Mr 330,000; domain structure A1-A2-B-A3-C1-C2) that is activated to factor Va (fVa; Mr 174,000) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2; Mr 94,000) and a light chain (A3-C1-C2; Mr 74,000). We analyzed the ultrastructure of scanning transmission electron microscope images of bovine and human fV, bovine fVa, and its constituent light chains and heavy chains. Factor V molecules had irregularly globular (10-12 nm) to oblong (8-14 nm) core structures which commonly displayed a peripheral satellite appendage of variable morphology attached to the core by a narrow stalk. Scanning transmission electron microscope mass analyses indicated that monomolecular bovine fV molecules had a mass of 322 +/- 45 kDa and human fV, 315 +/- 31 kDa. Factor Va molecules were irregular, globular (8-12 nm) structures that resembled the fV core structure, lacked the satellite appendage representing B domainal structures, and had a mass of 180 +/- 22 kDa. Our findings permit us to propose a structural model of fV suggesting the relative orientation of its closely associated light chain and heavy chain core components and indicating that these constituents remain associated in the transition from fV to fVa.

Animals↗

PubMed1990

Radioactive gold cluster immunoconjugates: potential agents for cancer therapy.

An 11 gold atom (undecagold) cluster was covalently attached to specific sites on Fab', F(ab')2 and whole IgG molecules such that each carried 11-33 gold atoms without significant loss of native immunospecificity. Gold cluster labeled 17-1A monoclonal F(ab')2 antibody fragments showed 80% immunoreactivity compared to native antibody fragments in binding to human colon carcinoma cells in vitro. Radioactive gold in vivo biodistributions in nude mice with human tumors are also reported. By using clusters, potentially a larger destructive payload can be carried per antibody.

Animals↗

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↗