PubMed Health⌕ Search

Biomedical subjects

M F Rohde

Publications and source records attributed to M F Rohde.

At least 55 records · Page 3Linked to original sources
PubMed1991

The effect of carbohydrate on the structure and stability of erythropoietin.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.

Animals↗
PubMed1991

Stoichiometric complexation of Streptomyces subtilisin inhibitor and subtilisin.

Subtilisin (Sbt) and Streptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.

Bacterial Proteins↗
PubMed1991

Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity.

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.

Arginine↗
PubMed1990

Cloning and nucleotide sequence of the N-acetylmuramidase M1-encoding gene from Streptomyces globisporus.

The gene (acm) encoding N-acetylmuramidase M1 (ACM) was cloned of Streptomyces globisporus ATCC No. 21553. The nucleotide sequence of the acm gene was determined and found to code for an ORF of 294 amino acids (aa). Comparison of aa sequence deduced from the acm gene with the N-terminal sequence of the extracellular enzyme suggests that ACM is synthesized with a 77-aa leader peptide. A comparison of the ACM aa sequence with the aa sequences of other proteins in the NBRF data base reveals that ACM has strong similarity to the N-O-diacetylmuramidase secreted by the fungus Chalaropsis.

Amino Acid Sequence↗
PubMed1990

Effect of amino-terminal processing by Staphylococcus aureus V-8 protease on activity and structure of recombinant human interferon-gamma.

Treatment of recombinant human interferon-gamma (rHuIFN-gamma) with Staphylococcus aureus V-8 protease generated a transiently stable species that lacks 10 amino-terminal residues. This protein showed distinct secondary and higher-order structures with an alpha-helical content of 31%, suggesting that the secondary and tertiary structure largely remain upon removal of 10 amino-terminal residues. The antiviral activity was abolished, or greatly reduced, for this species relative to the intact protein. These results suggest an important role for the amino-terminal portion in the activity of human interferon-gamma. Since the digested protein is difficult to refold from the acid-denatured state, it was concluded that, although not essential for maintaining the core structure of the protein, the amino-terminal portion is critical for refolding the protein from acid.

Amino Acid Sequence↗
PubMed1990

Alteration in folding efficiency and conformation of recombinant human tumor necrosis factor-alpha by replacing cysteines 69 and 101 with aspartic acid 69 and arginine 101.

An analog of human tumor necrosis factor-alpha (TNF-alpha) was created involving the replacement of Cys69 with Asp and Cys101 with Arg. The solution structure and behavior of this analog were compared with the native protein. The analog exhibited a greatly decreased folding efficiency following dilution from urea, but essentially identical circular dichroic spectra in both the folded and unfolded states. The Stokes radius of the native and analog TNF-alpha in the folded state were identical, with the analog exhibiting a slight broadening of the eluting peak. The fluorescence emission spectrum of the native protein exhibits a plateau from 320 to 328 nm, while the spectrum of the analog consisted of a single peak with a maximum at 335 nm. The analog also had a 1.4-fold increase in the fluorescence intensity. Limited proteolysis of the analog resulted in only one of the two peptides seen following digestion of the native protein, and this product was less stable than the equivalent native protein fragment. The analog exhibited a 10-fold lower cytolytic activity than the native protein. These results demonstrated that the disulfide bond is not necessary for folding and activity, but are consistent with the analog having a looser, more flexible structure in solution than the native TNF-alpha.

Amino Acid Sequence↗
PubMed1990

Characterization of a polyethylene glycol conjugate of recombinant human interferon-gamma.

Recombinant human interferon-gamma was conjugated with polyethylene glycol (PEG) using succinimidyl coupling of amino groups in the protein. The PEG conjugated material showed antiviral, growth inhibitory and macrophage activation activities indistinguishable from those of the unmodified protein. The PEG conjugation reduced the receptor binding affinity slightly, but increased the half-life of the protein when measured in rats. Almost no clearance was observed within 6 hr after injection for the PEG conjugated protein, whereas a rapid clearance was seen for the unmodified interferon-gamma. Two possible sites of PEG attachment were identified in the protein: the N-terminal amino group and either lysine 129 or 131.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber.

We have isolated the genomic and cDNA clones encoding a novel proteinase from the fungus Tritirachium album Limber, named proteinase T, synthesis of which is induced in skim milk medium. The coding sequence for this enzyme is interrupted by two introns in the fungal genome. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 53% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of two disulfide bonds, which might explain the thermal stability of the proteinase. We have expressed the proT cDNA in Escherichia coli. The authenticity of the product has been characterized by Western blotting and N-terminal analysis of the recombinant product.

Amino Acid Sequence↗
PubMed1989

Structure of bovine milk lipoprotein lipase.

The primary structure of bovine milk lipoprotein lipase (bLPL) was determined by alignment of peptides produced by tryptic digestion, Staphylococcus aureus V8 protease digestion, and cyanogen bromide cleavage. bLPL consists of 450 amino acid residues. Most tryptic peptides were isolated and analyzed, except for the dipeptide, Glu-Lys (position 423-424), and the 2 Lys at positions 416 and 488. Peptides resulting from digestion by S. aureus V8 protease and cyanogen bromide cleavage filled the missing part and completed the primary sequence of bLPL. The NH2 terminus of bLPL was determined to be Asp by sequencing the intact protein with a gas phase sequencer for up to 30 residues, whereas the COOH terminus was identified as Gly through, carboxyl peptidase Y cleavage. The enzyme contains 10 cysteine residues, all of which exist in disulfide linkages. They are formed between Cys29 and Cys42, Cys218 and Cys241, Cys266 and Cys285, Cys277, and Cys280, and Cys420 and Cys440. The sites of N-glycosylation were identified at Asn44 and Asn361. In accordance with a common structural homology of serine-type esterases, -G-X-S-X-G- (Yang, C. Y., Manoogian, D., Pao, Q., Lee, F., Knapp, R. D., Gotto, A. M., Jr., and Pownall, H. J. (1987) J. Biol. Chem., 262, 3086-3191), the active site serine of bLPL was assigned to the serine at position 134. The chymotrypsin nick of bLPL was determined to be between residues 390 and 391. A model of the enzyme is proposed on the basis of our data and available chemical data.

Amino Acid Sequence↗
PubMed1988

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine.

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.

Amino Acid Sequence↗
PubMed1986

Recombinant gamma interferon induces hypertriglyceridemia and inhibits post-heparin lipase activity in cancer patients.

Animals suffering from malignancy or chronic infection develop characteristic metabolic abnormalities, including a well-defined hypertriglyceridemic state. These abnormalities have been attributed to release of one or more mediators from activated macrophages. We report that cancer patients receiving RIFN-gamma, a potent macrophage activator, at doses of greater than or equal to 0.25 mg/m2/d i.m. show marked increases in triglyceride but not in cholesterol levels (pretreatment triglyceride level of 180 +/- 190 mg/dl [mean +/- SD] vs. a day-14 level of 370 +/- 242 mg/dl, n = 23, p less than 0.001 by the paired t test). This hypertriglyceridemia was characterized by an increase in very low-density lipoproteins and a decrease in plasma post-heparin lipase activity, consistent with defective triglyceride clearance (mean pretreatment lipase level of 2.1 mumol/ml/h vs. a day-14 level of 1.2 mumol/ml/h, n = 6, p = 0.02 by the paired t test). rIFN-gamma did not directly inhibit lipoprotein lipase enzymatic activity in vitro. Other possible mechanisms of action, such as suppression of lipase by an rIFN-gamma-induced mediator released from activated macrophages, or a direct effect of interferon on lipase biosynthesis, require further investigation. Our observations provide evidence that factors produced by the immune system can regulate lipid metabolism in man.

Adult↗
PubMed1985

Origin of apolipoprotein A-I polymorphism in plasma.

The origin and the functional significance of apo-A-I polymorphism in man has been investigated. Together with proapo-A-I (identified as A-I1 of the polymorphic series), four other isoforms are found in human plasma, namely A-I2, A-I3, A-I4, and A-I5. A-I3 is the "mature" product of proapo-A-I conversion in plasma. In this study we provide evidence that the other, more acidic, mature apo-A-I isoproteins are derived from A-I3 by a stepwise deamidation process. This conclusion is based on the following observations. 1) Incubation of A-I3 or A-I4, either free or associated with high density lipoprotein, produces a series of more acidic isoproteins corresponding to the sequence found in plasma. The conversion process fits in well with a first order reaction, and A-I3 to A-I4 conversion occurs virtually at the same rate as A-I4 to A-I5 conversion. 2) A-I3 and A-I4 have the same NH2- and C-terminal residues. 3) Formation of apo-A-I acidic isoproteins is accompanied by liberation of ammonia. In order to investigate whether deamidation of apo-A-I results in the production of forms which have different catabolism, a series of turnover studies was carried out in normal volunteers. A-I3 and A-I4 residence times in plasma were, respectively, 3.50 +/- 0.16 and 3.00 +/- 0.10 days (mean +/- S.E.; n = 3). Degradation rate of A-I3 was 8.81 +/- 0.69 mg/kg/day and that of A-I4 was 1.66 +/- 0.15 mg/kg/day (mean +/- S.E.; n = 3). Conversion of A-I3 to A-I4 and A-I4 to A-I5 occurred at the same rate in vivo as that observed in vitro. These results are consistent with the concept that A-I3 is the precursor to the other mature apo-A-I isoforms in plasma. A-I3 is the major isoform through which apo-A-I is eliminated from plasma.

Ammonia↗
PubMed1985

Conformational properties of human and rat apolipoprotein A-IV.

Apolipoprotein A-IV has been isolated from four sources: human and rat lymph and plasma. Conformational properties of the rat and human apoA-IV in solution and denaturation changes induced by guanidine hydrochloride (Gnd X HCl) were studied using circular dichroic and fluorescence spectroscopy, and analytical sedimentation equilibrium ultracentrifugation. We have shown that both rat and human apoA-IV have similar secondary structure with negative maxima in the circular dichroic spectra at 222 nm and 207 nm. Furthermore, we have found no significant difference in the alpha-helical content of the apoA-IV from rat plasma (33%), rat lymph (37%), human plasma (35%), or human lymph (35%). Our denaturation studies with Gnd X HCl demonstrated reversibility and the fact that each apoA-IV had a tendency to self-associate in solution and the self-association could be disrupted by low concentrations of Gnd X HCl (less than or equal to 0.4 M). Unfolding of the secondary structure of each apoA-IV occurred at higher concentrations of Gnd X HCl (midpoint less than or equal to 1.0 M). The apparent free energy of denaturation of the four apoA-IV proteins calculated from changes in the circular dichroic spectra upon addition of increasing concentrations of Gnd X HCl varied in a range from 3.0 to 4.2 kcal/mol. The fluorescence experiments revealed that apoA-IV from all sources had a maximum fluorescence emission at 342.5 nm, which shifted to the red region upon addition of increasing concentrations of Gnd X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1981

Physical properties of lipid-protein complexes formed by the interaction of dimyristoylphosphatidylcholine and human high-density apolipoprotein A-II.

Apolipoprotein A-II (apoA-II) from human plasma high-density lipoproteins associates with dimyristoylphosphatidylcholine (DMPC) to give complexes whose structure is determined by the temperature at which the reaction is conducted. The temperature dependence is related to the gel leads to liquid crystalline transition temperature, Tc, of DMPC which occurs at 23.9 degrees C. At T less than Tc (20 degrees C), T = Tc, and T greater than Tc (30 degrees C), three different complexes can be isolated. At 20 degrees C, at 75:1 (molar ratio of lipid to protein) complex is formed. This complex has a molecular weight (Mt) of 343 000, a Stokes radius, Rs, of 65 A, and a partial specific volume (v) of 0.914 mL/g. At 24 degrees C, two different complexes may be formed. One is similar to the one formed at 20 degrees C and the other is a complex with a DMPC:apoA-II ratio of 241:1; the corresponding physical constants for the latter complex are Mr = 1580 000, Rs = 120 A, and v = 0.948 mL/g. This complex is asymmetric, having a frictional coefficient f/f0 = 1.20. AT 30 degrees C, a 45:1 complex was formed; for this complex, Mr = 229 000, Rs = 57 A, and v = 0.892 mL/g. Electron microscopy reveals that the negatively stained complexes are arranged in rouleaux having subunits with average dimensions of 175 x 60, 250 x 62, and 50 x 55 A for the 45:1, 75:1, and 240:1 complexes, respectively. The multiple lipid-protein species formed by apoA-II and DMPC suggest the possible existence of more than one macromolecular spices of lipid and apoA-II in the plasma.

Apolipoprotein A-II↗
PubMed1980

Magnetic resonance studies of apolipoprotein C-I nitroxide labeled or [13C]methyl enriched at methionine-38.

One of the three proposed lipid-binding regions of the human apolipoprotein C-I (apo-C-I) is an amphipathic helix which extends from residue 33 to residue 53 and includes a single methionine at sequence position 38. The involvement of the sequence around methionine-38 in phospholipid binding has been evaluated with paramagnetic and nuclear reported groups attached to the thiomethyl moiety. This moiety has been spin-labeled with N-(2,2,6,6-tetramethylpiperidinyl-1-oxy)bromoacetamide or 13C enriched with 13CH3I. As determined from its EPR spectrum, the nitroxide at Met-38 of apoC-I had a rotational correlation time (tau C) of 0.22 ns. When dimyristoylphosphatidylcholine (DMPC) was bound to the spin-labeled apoprotein, tau c increased to 0.35 ns, indicating decreased motion for the methionyl side chain. The line width (nu 1/2) and spin--lattice relaxation time (T1) for the thiomethyl resonance of 13C-enriched apoC-I in 10 mM phosphate buffer was 6.0 Hz and 320 ms, respectively. When the protein solution was made 1.6 M in Gdn-HCl, these values changed to 2.6 Hz and 970 ms, respectively. Upon addition of DMPC multilamellar liposomes to [13C]apoC-I in 1.6 M Gdn-HCl, the line width increased to 4.7 Hz and the T1 decreased to 380 ms. These results strongly suggest that methionine-38 of apoC-I resides in a region of the apoprotein which undergoes significant secondary and/or tertiary structural change upon disaggregation/unfolding in Gdn-HCl and upon interaction with phospholipid.

Apolipoprotein C-I↗
PubMed1980

The conformational properties of human plasma apolipoprotein C-II. A spectroscopic study.

The solution properties of human plasma apolipoprotein C-II (apoC-II) have been studied by analytical ultracentrifugation, circular dichroic spectroscopy, and fluorescence spectroscopy. ApoC-II self-associates in solution, even though no rigorous thermodynamic analysis of the mode of self-association could be established. The reversible denaturation of apoC-II by guanidinium chloride (GdmCl) proceeded in a sequential fashion. Initial disruption of protein self-association by 0.3 M GdmCl was followed by cooperative unfolding of monomeric protein at higher GdmCl concentratioans with a midpoint 1.1 M GdmCl. Based on tryptophan fluorescence quenching unfolded apoC-II was more permeable to penetration by small molecules than the self-associated protein. a very low free energy (delta GH2O = 2.8 kcal/mol) of denaturation was calculated from the GdmCl denaturation titration curve. Heating of apoC-II to 55 degrees C did not induce a reversible cooperative unfolding of the protein. Calculations, bsed on Chou-Fasman probability algorithms, reveal three sequential helical regions in apoC-II and one beta sheet structure (residues 61-74P. The locations of these regions are consistent with the known physiological functions of apoC-II.

Amino Acid Sequence↗
PubMed1975

Molecular interactions between ribosomal proteins. Evidence for specificity of interaction between isolated proteins.

The proteins S2, S3, S5, and S10 from the 30S ribosomal subunit of Escherichia coli was studied by analytical ultracentrifugation to characterize them in solution and to determine whether isolated protein-protein interactions exist. Such interactions, if specific, may therefore bear some relationship to the spatial organization of the subunit structure. It was found that protein S2 self-associates to a slight extent and that solution mixtures of S2 and S3 contain only enough dimeric species to account for the S2 dimer. Hence, no observable interaction was detected between S2 and S3. Solution mixtures of proteins S5 and S10 revealed a species of molecular weight greater than either protein. The proposal is that S5 and S10 interact with an association equilibrium constant of 7.6 X 10(-5) M-1 at 3 degrees in a Tris buffer at pH 7.4. It was also shown that solution with a 1:1:1 mixture by mass, of S2, S5, and S10 contained a species possessing a molecular weight consistent with a simple ternary complex of the three proteins.

Bacterial Proteins↗