Sam Seifter

[22] Precipitation techniques

Publisher Summary This chapter describes methods for the precipitation of proteins for preparative purposes. Proteins can be precipitated by causing perturbations in the solvent with respect to pH, ionic strength, and temperature. The art and science of protein fractionation by differential solubility and precipitability has reached a pinnacle, and a wide variety of precipitation methods have been discovered empirically. Although some adsorbents, such as alumina and calcium phosphate gels were used, most of the methods employed several steps of ammonium sulfate precipitation. Perturbations that can cause various conformational transitions include (1) a rise in temperature that can weaken the strength of dipolar interactions, such as hydrogen bonds and can favor formation of hydrophobic interactions and (2) a decrease in temperature that can cause the reverse. Quantitative aspects of the resultant transitions in structure depend on the total numbers of specific kinds of interactions and variation in energies among individual interactions of the same kind. The inclusion of water-miscible solvents in the medium in which the protein is dissolved represents a considerable perturbation.

Collagen chain mRNAs in isolated heart cells from young and adult rats

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent. Fibroblast-like cells predominated in the non-myocyte cell fractions but endothelial and smooth muscle cells were also present. RNA from whole ventricular tissue served as a control. Northern and dot blot analyses were used to establish the presence or absence of mRNAs. In RNA prepared from whole ventricular tissue, the mRNAs for alpha-, beta-, and gamma-actin isotypes were detected whereas mRNA for alpha-actin was found in myocytes and those for beta- and gamma-actins were found in non-myocyte cells, confirming further the nature of the cell populations. Procollagen types I and III mRNAs were not detected in the total RNA of cardiomyocytes but mRNA for type IV collagen was present. The mRNAs for all three collagen types were present in the non-myocyte cells. These results suggest that in the rat heart the non-myocyte cells, probably fibroblasts, are responsible for interstitial collagen production. Both cell populations may engage in the formation of basement membrane collagen type IV.

Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization

Previous studies investigating the cellular origins of several collagens in young adult rat hearts (Eghbali et al., 1988) demonstrated that the mRNAs for types I and III collagen occurred in non-myocyte cells, mostly fibroblasts, whereas the mRNA for type IV collagen was observed in both myocytes and non-myocyte cells. In the present study, cellular localization of collagen mRNAs has been achieved by in situ hybridization in rat heart tissue and in isolated heart cells. Frozen tissue sections, isolated cardiomyocytes, cultured neonatal cardiomyocytes and fibroblasts were hybridized with DNA probes for type-specific collagens, actin, and myosin heavy chain. Silver grains were visualized by dark field imaging. In heart sections, types I and III mRNAs were observed predominantly adjacent to myocytes and in the interstitium, where fibroblasts are known to be present. In contrast, type IV collagen mRNA was identified both within the myocytes and the interstitium. In freshly isolated adult cardiomyocytes and in cultured neonatal cardiomyocytes, collagen type IV mRNA was observed but type I collagen mRNA was not. In cultured neonatal fibroblasts, both types IV and I collagen mRNAs were abundant.

Analysis for protein modifications and nonprotein cofactors.

Publisher Summary This chapter discusses protein modifications and non-protein cofactors. Numerous peptide hormones, hormone-releasing factors, and neurotransmitters are amidated at their carboxyl-terminal residues. Peptides in this group are generally synthesized with a glycine residue on the carboxyl terminus, as confirmed by the sequence of the specifying DNA or terminal codon of the mRNA. An indication that a given peptide undergoes α-amidation can be gleaned from comparison of the amino acid sequence deduced from coding nucleotide sequences with the amino acid sequence determined from analysis of the protein. The anchor is attached covalently to the carboxyl-terminal residue of the protein through an amide linkage with a phosphoethanolamine residue; it is noncovalently inserted into the membrane by means of the inositol phospholipid portion of the anchor. In contrast to the enzymes involved in chemotaxis that methylate L-glutamate residues, another class of enzymes known as “D-aspartate,” L-isoaspartate-methyltransferases catalyze the formation of methyl esters of D-aspartate and L-isoaspartate residues. The residual protein contains residues of diaminobutyric acid in place of methyl esters of glutamic acid and diaminopropionic acid in place of residues of aspartic acid.

Morphology, composition, and function of struts between cardiac myocytes of rat and hamster

SummaryThe morphology, composition, and function of struts that interconnect the lateral surfaces of cardiomyocytes were examined in the hearts of rats and hamsters. Methods included brightfield and fluorescent light microscopy, secondary and backscatter scanning electron microscopy, and transmission electron microscopy in conjunction with silver stain, cationic dye, and antibody to type-I collagen. These studies reveal a twisted, beaded appearance and a complex substructure of collagen fibrils embedded in a ground substance that has a positive reaction with cationic dye. A hierarchy of patterns of branching and attachment was seen among intercellular struts ranging in diameter from 0.1 μm to several urn. The hypothesis that struts tether not only the surfaces but the contractile lattices of laterally adjacent myocytes is supported by the following: (a) the attachments of struts to the collagen weave of the sarcolemma, often lateral to the level of Z bands, (b) the presence of collagen type I in a composite material arrangement, (c) the relative dispositions and configurational changes of struts and myocyte surfaces in various physiological states and induced, non-physiological perturbations of cardiac muscle, (d) the corrugated sarcolemmas with infoldings near Z bands, and (e) the continuity of intracellular filaments from Z bands to the inner aspect of the sarcolemma in relaxed and contracted myocytes. Implications of struts acting as tethers and sites for storage of energy in the motions of myocytes during the cardiac cycle are discussed.

Uptake of creatine by cultured cells

Abstract We have examined the uptake of creatine by cultured monolayers of human IMR-90 flbroblasts, human uterine smooth muscle cells, calf aortic smooth muscle cells, and myoblasts and myotubes of the L 6 E 9 rat skeletal muscle cell line. Creatine uptake is dependent on temperature and sensitive to the presence of Na + in the extracellular medium. It is saturable, apparently concentrative, and inhibited by ouabain and structural analogs of creatine. In these respects, it resembles the process of creatine uptake by isolated preparations of skeletal muscle and brain tissues. Lineweaver-Burk plots of the data for variation in rate of uptake with concentration of creatine in the medium are nonlinear, suggesting that the process of uptake may be heterogeneous. Assuming the operation of two saturable processes of uptake, we calculated two values for apparent K m and V for each cell line. Kinetic parameters of creatine uptake by the different cell types are similar. The lower values of K m (0.02–0.04 m m ) are in the physiological range of creatine concentration in mammalian plasma.

2 Biochemistry and molecular biology of MNSs blood group antigens

: This chapter has reviewed the nature of antigens of the MNSs blood group system. The structures of the proteins and the molecular features and organization of glycophorin genes were described, emphasizing their domain arrangement and the extensive sequence homology that indicates that their common and variant alleles belong to a single gene family. Methods currently used to examine these antigens are immunoblotting and DNA typing. The majority of variant genes are hybrids of parent glycophorin genes in a variety of arrangements; they contain no other sequences but those of the parent genes. The structures of the hybrids are summarized in Figure 8. Several hybrids appear to have arisen by unequal homologous recombination but others appear to have occurred through gene conversion. In this system the molecular genetic basis for a single variant phenotype may differ, as documented by gene rearrangements that appear to have occurred, as separate events, at different sites in the same intron; this has resulted in protein structures (hence phenotypes) that are identical. For example, unequal homologous recombination occurring within intron 3 can have given rise to only a limited number of phenotypes, namely alpha M-delta S, alpha N-delta S, alpha M-delta S, alpha N-delta S and delta-alpha. In addition, different sites of an exon may have been involved in gene rearrangements through gene conversion leading to nearly identical protein structures, yet different serological phenotypes. Thus, gene conversion could be more significant for generation of antigenic diversification as the number of possible new alleles is quite large. The participation of the HGpE gene in these rearrangements would make this number even larger. New sites and the expressed pseudoexon have created the epitopes of the variant phenotypes, and sequences specific for several variant antisera have been identified. Thus, the molecular basis for several serological reactions involving this system is now better understood.

18 The Collagenases

Publisher Summary This chapter describes the collagenases of Clostridium histolyticum , the collagenase of the tadpole of Rana catesbiana , and the collagenases of human rheumatoid synovial fluid. A collagenase is defined as an enzyme capable of causing the hydrolytic scission of peptide bonds located in the characteristic poly-L-proline type of helical regions when the substrate is in the undenatured state. Almost all of the collagenases are inhibited by ethylenediaminetetraacetate (EDTA) and of these all except that of Trichophyton schoenleinii appear to require Ca 2+ for activity. Several collagenases are inhibited by cysteine, and, of these, a group appears to contain zinc or some other metal in their structures. However, there are both bacterial and tissue collagenases that appear not to be inhibited by cysteine. The laboratory applications of collagenases have been limited largely to clostridiopeptidase A. Clostridial collagenases have been employed to study the crystalline regions of the collagen molecule and also to provide fragments for study from the amorphous regions. Clostridiopeptidase A has been used effectively to study the unfolding and refolding of the collagen helix.

Glucocorticoid receptors in WI-38 fibroblasts Characterization and changes with population doubling in culture

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoids was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone or 17 beta-estradiol. WI-38 cells exposed to [3H]dexamethasone at 30 degrees C were able to transfer the 3H-labeled steroid-receptor complex to the nuclear materal. A reduction of 30--50% was observed in the number of [3H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [3H]dexamethasone as a function of the two passage levels were detected.

Properties of heart fibroblasts of adult rats in culture

SummaryIn the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.