Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

The effects of fetal exposure to 1,2-dibromo-3-chloropropane on adult male reproductive function

Several drugs have been shown to cross the placental barrier and affect the fetal testis causing a reduction in testosterone with a resultant impairment of sexual differentiation and an ultimate problem in adult sexual function. In this study, pregnant female rats were treated with 25 mg/kg of the pesticide 1,2-dibromo-3-chloropropane (DBCP). Treatment began on Days 14.5, 16.5, or 18.5 and continued through Day 19.5 of gestation. Some animals were killed on Day 20.5 of intrauterine life and fetal intratesticular testosterone was measured. All other animals were allowed to deliver, and the males were raised to adulthood. At adulthood, body, testis, prostate glands and seminal vesicle weights were recorded. Intratesticular testosterone and luteinizing hormone (LH) receptors were measured. Male and female sexual behavior was quantified and the volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus was calculated. The histological appearance of the testis was also examined. Treatment for 6 days during fetal life with DBCP decreased intratesticular testosterone by 50% compared to controls at 20.5 days of gestation. At adulthood, all male rats treated during fetal life had a reduced body weight that was correlated with the duration of exposure. Adult testis weight was reduced to 75% of controls as a result of 2 days of fetal exposure to DBCP, whereas 4 and 6 days of exposure during fetal life reduced testis weight by greater than 90%. LH receptors and intratesticular testosterone, in the adults treated during fetal life, were also dramatically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride

The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 degrees C. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four species in the reaction.     

Partial characterization of the phosphotransferase system of human central-nervous-system myelin.

The phosphotransferase system of human central-nervous-system myelin was investigated. Evidence obtained indicated the presence of at least two different phosphotransferase systems (cyclic nucleotide-dependent and -independent) in myelin, which were found to be firmly associated with the membrane. The cyclic AMP-dependent kinase of myelin and white-matter cytosol preferentially phosphorylated certain histone fractions and displayed only modest activity with basic protein as substrate. On the other hand, the cyclic nucleotide-independent system showed specificity toward basic protein. Its activity was not only dependent on Mg2+ but it was greatly enhanced by this bivalent cation. Whereas the cyclic nucleotide-dependent kinase could be extracted with buffers containing Triton X-100, the bivalent cation-regulated kinase resisted solubilization from myelin under these conditions.     

Fluorouracil and the isolation of mutants lacking uridine phosphorylase in Escherichia coli: location of the gene

Summary A selective technique is described for the isolation of mutants of Escherichia coli lacking uridine phosphorylase and the location of the gene specifying this enzyme on the bacterial chromosome is determined. Using strains with appropriate lesions it is shown that there are three routes via which 5-fluorouracil can be converted to compounds which inhibit cell growth.     

On the carbohydrate—protein linkage group in vitreous humor hyaluronate

Fractional molar ratios of serine, threonine and aspartic acid to neutral sugars in the purified bovine vitreous humor hyaluronate, and a 4–5-fold increase in the percentage of these amino acids and the absence of sugar alditols in hyaluronate reduced with NaBH₄---PdCl₂ after alkali treatment indicated the absence of a carbohydrate—protein linkage. Gel filtration behavior, a decrease in intrinsic viscosity of reduced hyaluronate to about one-half and a significant decrease in its specific rotation suggested that the two antiparallel chains of the hyaluronate double helix may come apart upo reduction. The vitreous humor hyaluronate contained 109.2 ppm of “bound” silicon. It is suggested that the bound silicon may bridge the two antiparallel chains through the neutral sugars and/or through the hydroxyl group of the uronic acid moiety.     

Intra- and extraganglionic peripheral cholinergic neurons in the urogenital organs of the cat

Summary The distribution of cholinergic neurons in the urinary tract and male genital organs of the cat was studied by a histochemical method for acetylcholinesterase. In addition to cell clusters in autonomic ganglia (intraganglionic cells), isolated extraganglionic cholinergic cells were found within the innervated tissues, usually in association with nerve trunks and blood vessels. Smaller neural cells with multiple axonal processes, identical to Cajal's interstitial cells, were found in the meshes of the terminal nerve plexus in smooth muscle, lamina propria and vascular wall.It is concluded that peripheral cholinergic neurons, like their adrenergic analogues, are arranged as a short intraganglionic, a shorter extraganglionic, and a terminal system of neurons.Supported in part by grants 10465 and 11285 from the USPHS and the Henry C. Buswell Urology Research Fund.     

Results of skin test surveys for systemic Mycoses in Iran

Several investigators had carried out histoplasmin skin test surveys in 3 different areas of Iran before 1960 (5). To detect skin sensitivity to both histoplasmin and coccidioidin a study was carried out by the Institute of Public Health Research in parts of 7 of the country's 13 provinces on both apparently healthy persons and on clinic patients from February 1962–September 1966. These studies uncovered a small focus with a rather high level of positive reaction to histoplasmin (about 17 %). Positive cultures ofHistoplasma capsulatum were not obtained from either sputum of the positive reactors or from collected soils. In spite of a previously reported case of coccidioidomycosis, very low percentages of positive reaction to coccidioidin were noted.These studies were supported in part by the Institute of Public Health Research Teheran University, and funds of the Ministry of Health and Plan Organization for Project No. 631101 and N.I.H. Grant No. TW00170.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).