Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Surgical correction of Stahl's ear using the cartilage turnover and rotation method

Various operative methods for the correction of Stahl's ear exist. We, however, have devised an alternative "turnover and rotation" procedure. In this procedure, the deformed cartilage itself is used in its original contour to form a new scaphoid fossa, thus simplifying the corrective procedure and establishing a reliable operative method in which reversion to the original state is thought to be highly unlikely.     

Reevaluation of the amino acid sequence of porcine follitropin

We have reevaluated the sequence of porcine follicle-stimulating hormone (pFSH) with more recent protein-sequencing methodology. This has led to revision of the earlier proposed sequence. As with almost all reported gonadotropin -subunits, NH₂-terminal heterogeneity was found in the porcine FSH -subunit (FSH), starting with residue Phe (1), Asp (3), Gly (4), or Thr (7). In the -subunit, there were found to be at least two molecular species, starting with residue Asn (1) (minor 20%) or Cys (3) (major 80%) as NH₂-terminal and ending at residue Glu (108) as COOH-terminal. The net effect of the present revisions is to increase the homology of pFSH with other reported follitropin sequences. Apparent differences in the half-cystine placements in a previous proposal for pFSH compared with other species of FSH are no longer tenable. The half-cystine placements thus remain a constant structural feature throughout the gonadotropin hormones (choriogonadotropin, follitropin, and lutropin).     

Our method of correcting cryptotia

Our technique for the correction of cryptotia using both Z-plasty and the advancement flap is described. The main advantages are the simple design of the skin incision and the possibility of its application to cryptotia other than severe cartilage deformity and extreme lack of skin.     

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha.

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.     

Physicochemical characteristics of human sex hormone binding globulin: evidence for two identical subunits

We have developed a rapid protocol for the purification of human sex hormone binding globulin (SHBG) which allows the protein to be purified from pregnancy serum within 48 h. This minimizes any possible degradation of the protein by serum proteases, and has enabled us to re-examine some important and controversial aspects of its structural composition. Our physicochemical data are consistent with the hypothesis that SHBG is a dimeric glycoprotein composed of 2 protomers that exhibit size heterogeneity (approximately 50 and approximately 52 K daltons). The dimeric SHBG molecule appears to contain only approximately 8% carbohydrate, and sequence information indicates that an N-linked oligosaccharide chain may be attached to residue 7 (asparagine) from the NH2-terminal amino acid (leucine). When compared with earlier reports, differences in the relative amounts of heavy (approximately 52 K) and light (approximately 50 K) protomers, and the microheterogeneity of NH2-terminal amino acids, have led us to conclude that they may be caused by proteolytic degradation in vivo as well as during the storage of blood samples prior to protein purification. However, the NH2-terminal amino acid sequence data indicate that the primary structures of the heavy protomers, which evidently interact to form the majority of SHBG dimer in serum, are similar and may even be identical. Evidence to support this is provided by the observation that a monoclonal antibody, which recognises a configurational epitope, interacts with two epitopes per native dimeric form of human SHBG.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.