Neocarzinostatin: spectral characterization and separation of a non-protein chromophore.

Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.     

Neocarzinostatin: Chemical characterization and partial structure of the non-protein chromophore

The molecular formula C₃₅H₃₅NO₁₂ (mol.wt. 661) is proposed for the biologically active chromophoric component of neocarzinostatin. The partial structure $\text{2}$ is proposed based on ${}^1\text{H}$ NMR and mass spectral data and consists, in part, of a 2,6-dideoxy-2-methylamino-galactose moiety and a naphthoic acid derivative. Special treatments required to obtain spectral data of the labile chromophore are described.     

Purification and characterization of a protein containing D-aspartic acid in bovine lens

A protein containing biologically uncommon D-aspartic acid (DAsp) was extracted with 60% EtOH from the water-insoluble fraction of bovine lens. The protein was purified by DEAE-TOYOPEARL chromatography and electrical elution by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by reverse-phase chromatography. The D/L ratio of aspartic acid in the protein isolated was 0.12. The molecular weight of this protein was estimated to be 22,500 by SDS-PAGE. The high content of serine, glycine and glutamic acid was noteworthy. It has been considered that the presence of DAsp in the living body is caused by racemization closely related to aging. The age of bovines used was relatively young (5 years old). If the racemization was caused by aging, the presence of DAsp in the relatively young bovine lens suggested that the aging of the lens protein may start at a relatively young age. The protein containing DAsp may be generally present in lens beyond species such as mouse, bovine and human.     

Purification and characterization of protein IIIb, a mammalian brain phosphoprotein

The phosphorylation of a 55,000-dalton protein (Protein IIIb) present in mammalian brain was previously shown to be increased by depolarizing agents in the presence of calcium, by cyclic nucleotides, and by appropriate neurotransmitters. We now report that Protein IIIb has been purified 660-fold to near homogeneity and partially characterized. The hydrodynamic properties of the purified protein indicate that it exists as an elongated monomer. cAMP-dependent protein kinase catalyzes the incorporation of 0.82 mol of phosphate into serine/mol of protein. The protein is heterogeneous in isoelectric focusing, exhibiting multiple forms with isoelectric points ranging in pH from 6.6 to 7.3.     

Structural characterization of a thiazoline-containing chromophore in an orange fluorescent protein, monomeric Kusabira Orange

Monomeric Kusabira Orange (mKO) is a green fluorescent protein (GFP)-like protein that emits orange light at a peak of 559 nm. We analyzed its X-ray structure at 1.65 A and found a novel three-ring chromophore that developed autocatalytically from a Cys65-Tyr66-Glu67 tripeptide in which the side chain of Cys65 formed the third 2-hydroxy-3-thiazoline ring. As a result, the chromophore contained the CNCOH group at the 2-position of the imidazolinone moiety such that the conjugated pi-electron system of the chromophore was more extended than that of GFP but less extended than that of the Discosoma sp. red fluorescent protein (DsRed). Since a sulfur atom has potent nucleophilic character, the third 3-thiazoline ring is rapidly and completely cyclized. Furthermore, our structure reveals the presence of a pi-pi stacking interaction between His197 and the chromophore as well as a pi-cation interaction between Arg69 and the chromophore. These structural findings are sufficient to account for the orange emission, pH tolerance, and photostability of mKO.     

Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a

The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with (125)I-plasminogen. A 54-kDa protein lost the ability to bind (125)I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human (125)I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by epsilon-aminocaproic acid. A single class of binding sites was detected, and a K(d) of 0.57 +/- 0.14 microm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.     

Purification, characterization, and structural analysis of a plant low-temperature-induced protein.

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.     

Purification and characterization of an inactive form of cAMP-dependent protein kinase containing bound cAMP

By a new procedure, the holoenzyme of bovine heart type II cAMP-dependent protein kinase was purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A high performance liquid chromatography-DEAE purification step resolved two distinct peaks of protein kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. The two peaks exhibited similar Stokes radii and sedimentation coefficients. They had similar ratios of regulatory to catalytic subunits both by densitometric scanning of SDS-PAGE bands and by the ratios of equilibrium [3H]cAMP binding to maximal kinase activity. These results suggested that the holoenzyme of each peak contained two regulatory subunits and two catalytic subunits, although a subpopulation of holoenzyme lacking one catalytic subunit also appeared to be present in Peak 2. Assays of cAMP indicated that the Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Determination of [3H]cAMP dissociation rates showed that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP. Interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2. Peak 2 holoenzyme, as compared to Peak 1, had enhanced binding in nonequilibrium [3H]cIMP and [3H]cAMP binding assays, as was expected due to the presence of cAMP and to the known positive cooperativity in binding of cyclic nucleotides to the kinase. The positive cooperativity in kinase activation, as indicated by the Hill coefficient, was greater for Peak 2 than Peak 1, but the cAMP concentration required for half-maximal activation (Ka) of each of the two peaks was very similar. In conclusion, Peak 2 is an inactive ternary complex of cAMP, regulatory subunit, and catalytic subunit, and Peak 1 is a cAMP-free holoenzyme. The cAMP-bound form may represent a major cellular form of the enzyme which is primed for activation.     

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endoPGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.     

beta-actinin, a regulatory protein of muscle. Purification, characterization and function.

beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.     

Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.     

Purification and characterization of yeast Sco1p, a mitochondrial copper protein

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains approximately 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.     

Purification and characterization of proteoliaisin, a coordinating protein in fertilization envelope assembly

We report the purification and characterization of proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope. Proteoliaisin was purified from egg cortical granule exudate to greater than 99% homogeneity using chromatography on DEAE-Sepharose and on phenyl-Sepharose. Native proteoliaisin is a highly asymmetric protein (f/fo = 2.0) composed of a single Mr approximately 230,000 peptide. Its asymmetry was demonstrated both by analytical ultracentrifugation and by nondenaturing polyacrylamide gel electrophoresis, a novel analysis that detects molecular asymmetry in heterogeneous protein mixtures. Proteoliaisin is enriched in six amino acids: aspartic acid/asparagine, glutamic acid/glutamine, glycine, and cysteine, which account for over 50% of its mass. Nearly all of the cysteine residues are disulfide bonded. The protein contains a small proportion of aromatic amino acids with phenylalanine greater than tyrosine greater than tryptophan. At neutral pH its absorbance maximum is at 274.5 nm, with an extinction coefficient of 0.43 ml mg-1 cm-1. Proteoliaisin forms a 1:1 Ca2+-stabilized complex with ovoperoxidase, another component of the fertilization envelope, with Kd = 1.1 X 10(-6) M. Proteoliaisin, a constituent of the specialized echinoderm extracellular matrix called the fertilization envelope, has certain structural similarities to mammalian extracellular matrix proteins.     

Examination of a nerve injury-induced, 37 kDa protein: Purification and characterization

Following traumatic injury to the adult rat sciatic nerve the synthesis and accumulation of soluble, extra-cellular, 37 kDa protein is increased. This protein, which accumulates in the extracellular space of the injured nerve, accounts for nearly 5% of the total soluble pool of protein in an injured nerve 3 weeks after injury. 8 weeks after injury, when regeneration is nearly complete, this accumulated pool returns to control levels, yet if regeneration is blocked synthesis of the 37 kDa protein remains high. Recently this 37 kDa protein has been shown to be nearly identical to apolipoprotein E, the protein component of various lipoprotein particles. This finding suggests a role for the 37 kDa protein in cholesterol and lipid transport and metabolism during nerve repair within the nervous system, functions that have been ascribed to apo E in serum. Results are presented here describing the purification of the nerve injury induced 37 kDa protein and the subsequent production of specific rabbit antisera directed against it. By centrifugation analysis in a sucrose gradient, a native mass of 37 kDa was determined, revealing the 37 kDa protein's monomeric, native structure. Additionally injections of [³⁵S]methionine directly into the injured nerve allowed 1) a comparison of 37 kDa synthesis in vivo versus in vitro and 2) an examination of the presence or absence of retrogradely transported 37 kDa protein. The in vitro and in vivo collected material were found to share identical 2-dimensional electrophoretic mobilities, and no appreciable amount of transported 37 kDa protein was found in proximal regions of the injured nerve.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Purification and characterization of an immunomodulatory endometrial protein, glycodelin

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.     

Purification and characterization of a protein kinase from pine pollen

A kinase phosphorylating casein and phosvitin has been purified from pine pollen by a three-step procedure involving DEAE-cellulose chromatography, affinity chromatography on casein-Sepharose and Sephadex G-100. A purification of about 2000 fold was obtained by this procedure. The kinase is affected neither by cyclic nucleotides nor by Ca²⁺-calmodulin, whereas it is strongly inhibited by heparin. Using this purification procedure, we have isolated protein kinase exhibiting phosphorylating activity towards casein in the pollen of many other Pinaceae species.     

Purification and characterization of a trypsin-like protein from rat pancreas

The purification of the latent form of a rat pancreas trypsin-like protein was performed by ion-exchange and hydrophobic chromatographies. After partial activation, the affinity on immobilized soybean trypsin inhibitor allowed the isolation of an active and an inactive form. They had 30,000 and 32,000 molecular weight, respectively, as checked by polyacrylamide slab gel electrophoresis. Active enzyme (named TLP) was not glycosylated and had an isoelectric point of 4.4. The rate of hydrolysis of different substrates and the effects of various proteinase inhibitors indicated clearly that TLP differs from proteinases previously described and belongs to the trypsin family.