Site-directed mutagenesis defines a domain in the gonadotropin alpha- subunit required for assembly with the chorionic gonadotropin beta- subunit

1992 ◽  
Vol 6 (2) ◽  
pp. 261-271 ◽  
Author(s):  
M. Bielinska
Keyword(s):  
Chorionic Gonadotropin ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Directed Mutagenesis

scholarly journals Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the α- and β-subunits

1989 ◽  
Vol 264 (1) ◽  
pp. 257-264 ◽  
Author(s):  
H M Kent ◽  
I Ioannidis ◽  
C Gormal ◽  
B E Smith ◽  
M Buck
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Component ◽  
Molybdenum Cofactor ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Diazotrophic Growth ◽  
Iron Molybdenum Cofactor

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

scholarly journals Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon

1989 ◽  
Vol 260 (3) ◽  
pp. 737-747 ◽  
Author(s):  
D Buck ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Binding Site ◽  
Phosphorylation Site ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Directed Mutagenesis ◽  
Oxoglutarate Dehydrogenase ◽  
Succinyl Coa Synthetase

The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.

Discordant Results in Human Chorionic Gonadotropin Assays

1992 ◽  
Vol 38 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Laurence A Cole ◽  
Andrew Kardana
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Terminal Segment ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Individual Sample ◽  
Serum Samples ◽  
Subunit C ◽  
Core Fragment ◽  
Alpha Type

Abstract Discordance has been reported in human chorionic gonadotropin (hCG) concentrations measured by different immunoassay kits. We examined the results for 40 serum samples assayed with 10 different hCG immunoassay kits. Results varied considerably. Individual sample results varied by as much as 58-fold. Average results for different kits varied by as much as 1.4-fold for pregnancy (20 samples) and 2.2-fold for trophoblast disease (20 samples) serum. We investigated the causes of this discordance. hCG or hCG beta are general names for mixtures of hCG, hCG alpha, or hCG beta immunoreactive molecules in serum. These mixtures include regular hCG, nicked hCG (missing peptide linkages at beta 44-45 or beta 47-48), carbohydrate variants of hCG, hCG missing the beta-subunit C-terminal segment, free beta-subunit, beta-core fragment, and free alpha-subunit. We prepared standards for each of these major variants and measured their reactivities in the 10 hCG immunoassay kits. Free beta-subunit reactivity varied from nonrecognition (anti-beta:anti-alpha type kits; Hybritech Tandem-R and others) to overrecognition (one kit had five-fold greater affinity for free beta than for hCG). Kits with antibodies to beta-subunit C-terminal segment (Organon NML and others) failed to recognize hCG missing this segment, a component of serum hCG in trophoblast disease. Kits with anti-hCG antibodies (Serono MAIA-clone and others) had minimal recognition of nicked hCG (12%), a component of all serum hCG samples, and consistently gave the lowest values with all serum samples. We conclude that differences in recognition of nicked hCG, free beta, and these other hCG variants cause discordance in hCG immunoassay results.

Free α-Subunit, Free β-Subunit of Human Chorionic Gonadotropin (hCG), and Intact hCG in Sera of Healthy Individuals and Testicular Cancer Patients

1992 ◽  
Vol 38 (3) ◽  
pp. 370-376 ◽  
Author(s):  
S Madersbacher ◽  
R Klieber ◽  
K Mann ◽  
C Marth ◽  
M Tabarelli ◽  
...  
Keyword(s):  
Testicular Cancer ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Serum Concentrations ◽  
Healthy Individuals ◽  
Pathological Conditions

Abstract To determine the serum concentrations of human chorionic gonadotropin (hCG), its free beta-subunit (hCG beta), and the free alpha-subunit (free alpha) common to all human glycoprotein hormones under physiological and pathological conditions, we developed monoclonal antibody-based immunoenzymometric assays. Free alpha-subunit was detected in the sera of all healthy individuals of both sexes; hCG was measurable in sera of 54% of the men, and 46% were positive for free hCG beta; in nonpregnant women, 69.5% were positive for hCG, 68.4% for the free beta-subunit. Pathological conditions, i.e., hCG-producing tumors, were studied in vitro and in vivo. In vitro, the concentrations of hCG, free hCG beta, and free alpha in tissue-culture supernates of a choriocarcinoma cell-line ("JAR") showed a parallel pattern during time-course analysis. In vivo, in long-term follow-up studies of 13 patients with testicular cancer, serum concentrations of the three analytes paralleled each other, whether the disease was in remission or not. Because of a selective increase of free hCG beta and free alpha in 27% of seminomatous tumor patients and in 13% of the nonseminomatous patients, the percentage of tumor-marker-positive sera was increased from 15% to 42% and 57% to 70%, respectively, by the additional measurement of free hCG beta and free alpha. Thus hCG, free hCG beta, and free alpha are physiologically present in a high percentage of the sera from healthy men, and the determination of free hCG beta and free alpha, although not of prognostic value, improves the diagnostic possibilities in patients with testicular cancer.

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