Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.     

Mitochondrial uncoupling protein 1 expression in thymocytes

Using an antibody specific and selective to mitochondrial uncoupling protein 1 (UCP1) peptide, this study confirms the observation that UCP 1 is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.     

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

Subtilisin Protein Inhibitor from Potato Tubers

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.     

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

The effect of thyroxine on the calmodulin-dependent (Ca2+-Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin-dependent (Ca2+-Mg2+)ATPase and the activity of this enzyme as well as that of (Na+-K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+-K+)ATPase, while the activity of calmodulin-dependent (Ca2+-Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP-dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP-independent phosphorylation of several other peptides was augmented. Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP-independent protein kinase(s) and lower activity of cAMP-dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin-dependent (Ca2+-Mg2+)ATPase protein and affects the activity of cAMP-independent and cAMP-dependent protein kinases bound to sarcolemma.     

Isolation of Three Crystalline Proteins from Beef Liver After Partial Purification by Acetone Fractionation

The isolation of three proteins in crystalline form from ground beef liver is described. These proteins are FTBL protein (Arch. Biochem. Biophys. 188, 251–265 (1978), crotonase, and catalase. Crotonase is isolated by crystallization from a 32 acetone extract of the ground liver. FTBL protein and catalase can subsequently be isolated from the same extract. For optimal yield and ease of isolation, FTBL protein is isolated from a 46.5% acetone extract from which catalase can subsequently be crystallized by dialysis.

The isolation of FTBL protein as well as the isolation of catalase involves a preliminary fractional precipitation and solution before crystallization can be achieved. Isopropanol can be substituted for acetone in the isolation of the above three proteins and in the case of catalase, results in an exceptionally high yield.

Methods for the recrystallization of the proteins are presented and the role of organic solvents in recrystallization is discussed.     

Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.     

Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.     

Biochemical characteristics of functionally active actin-like protein from liver mitochondria

The actin-like protein with a molecular weight of 42 kDa was obtained from the preparation of freshly isolated mitochondria of the rat liver using the method of immobilized DNAse affinity chromatography. The inhibitory ability of the isolated protein with respect to pancreatic DNAse I was the same as that of muscular actin. The native structure of the mitochondria protein is confirmed by the data of spectral analysis and its ability to globular-fibrillar transformation with an increased ionic strength of the solution. The polymerization ability as well as a stimulating effect of the actin-like protein of mitochondria on the ATPase activity of myosin is much less pronounced as compared to actin of skeletal muscles.     

Postmortem Accumulation of Tubulin in Postsynaptic Density Preparations

Abstract: Postsynaptic density (PSD) preparations isolated from canine cerebral cortex that had been left at 0–37°C for various times were found to become enriched in two bands in a time- but not temperature-dependent manner. The two bands were identified as tubulin subunits by gel mobility and immunology. Of all the isolated synaptic structures the increase in tubulin occurred primarily in the PSD fraction. The increase of tubulin also occurred in PSD preparations isolated from canine cerebellum and rat forebrain. Results obtained when PSD fractions were isolated from canine brain obtained as rapidly as possible after the death of the animal indicate that the maximum amount of tubulin in the PSD preparations is 2.5% of total Coomassie blue-stained protein as determined by scanning of gel electrophoretograms. These results imply that tubulin is probably not a major structural protein of the PSD as it exists in situ.     

The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.

A family of eukaryotic RNA binding proteins is defined by the conserved RNP motif. The poly(A) binding protein has four such motifs. We report on the isolation and structural characterization of several variant cDNA clones, as well as of a gene encoding this protein in Xenopus laevis embryos. Wild-type protein as well as truncated versions carrying isolated single motifs or artificial combinations of two and more such elements were characterized for their ability to bind specifically to RNA homopolymers. Three of the isolated repeats were functional in specific RNA binding, whereas the N-terminal RNP motif was non-functional. Combinatorial effects in RNA binding were measured with constructs carrying multiple repeats, which were not predictable from the activity of isolated domains.     

Isolation and initial characterization of the mammalian midbody

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.     

Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. β-ν-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.     

Proteins from the lichenXanthoria parietina which bind to phycobiont cell walls. Correlation between binding patterns and cell wall cytochemistry

Summary A protein fraction was isolated from the lichenXanthoria parietina which bound to the appropriate cultured phycobiont, but not to the freshly isolated symbiotic alga. The protein also appeared to discriminate between five other strains of cultured phycobionts from different lichens; phycobionts isolated from lichens in the familyTeloschistaceae bound the protein whereas phycobionts isolated from lichens in other families did not. Using cytochemical techniques, it was shown that protein binding ability was correlated with high levels of acidic polysaccharide in the cell wall, and the presence of a protein coat on the cell wall surface of the phycobiont. The possible role of this protein in recognition between lichen symbionts is briefly discussed.     

Photodynamic action and bacterial protein synthesis

Summary Photodynamic treatment of bacterial cells with thiopyronin and psoralen inhibits protein synthesis. Transfer RNA isolated from thiopyronin- and psoralen-treated cells is inactive in cell-free protein synthesis. Both the sensitizers inactivate transfer RNA to about 60 to 70%. Ribosomes isolated from thiopyronin-sensitized cells lose 45% of their activity, whereas under the influence of psoralen only 20% inactivation is observed. The enzyme fraction is not damaged in psoralen-treated bacteria, but thiopyronin inactivates this fraction as well. In vivo experiments indicate that psoralen does not react with the protein components of the cell. The mechanism of these inactivations is discussed.