Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions.

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.     

Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.

A strain of Escherichia coli K-12 has been isolated that carries a Mu bacteriophage-induced mutation in the ent gene cluster. Nutritional tests together with examination of the compounds accumulated by the mutant strain indicated that the mutant was blocked both in the synthesis of 2,3-dihydroxy-benzoate and its subsequent conversion into enterochelin. Enzymic complementation assays of the mutant with several mutants each affected in one of the ent genes showed that the Mu-induced mutant was entA-, entB-, entC+, entD+, entE+, and entF+. Since the mutant produced the entD, entE, and entF gene products but was unable to produce enterochelin from 2,3-dihydroxybenzoate, it must therefore be affected in an additional protein concerned with this conversion. It is therefore postulated that the Mu-induced mutation affects a previously unrecognized gene, entG. Genetic experiments indicate that the mutation in strain AN462 which affects the three ent genes is the result of a single insertion of Mu in the ent gene cluster. This polarity mutant therefore provides evidence that three of the ent genes are part of an operon.     

Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3–4F cells are unable to utilize nicotinic acid. When 3T3–4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the fourday period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.     

7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.     

Molecular size of nerve growth factor in dilute solution.

Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known.     

Kinetics of mucopolysaccharide and glycoprotein synthesis by chick embryo chondrocytes. Effect of D-glucose concentration in the culture medium.

Late log cultures of chick embryo vertebral chondrocytes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum consume D-glucose from the culture medium at a rate of approximately 0.40 mumol per h per 10(6) cells. When the D-glucose concentration in the medium drops below 1 mumol per ml the glycogen stores are rapidly exhausted, and cell growth ceases. 35SO4(2)- is incorporated into chondroitin-6-SO4 and chondroitin-4-SO4 at linear rates of 1.2 and 0.4 nmol per h per 10(6) cells, respectively, until the D-glucose level in the medium drops below 1 mumol per ml, but there is always a slight lag in the initial appearance of chondroitin-4-SO4. Throughout the period of 35SO4 2- labeling, the amount of chondroitin-6-SO4 that is recovered in the cells exceeds the amount that is recovered in the medium, but the opposite is true for chondroitin-4-SO4. However, when cells prelabeled with 35SO4(2-) are then transferred to a label-free medium, the secretion of chondroitin sulfates proceeds at much slower rates, and the kinetics of chondroitin-6-SO4 and chondroitin-4-SO4 secretion are very similar. In this chase experiment the chondroitin sulfates are recovered quantitatively after a 24-h incubation period, indicating that these embryonic chondrocytes do not degrade the chondroitin sulfates under these culture conditions. The rate of incorporation of counts from D-[14C]glucosamine into mucopolysaccharides and glycoproteins increase with time. This nonlinear rate results from a progressive increase in the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool as the D-glucose in the culture medium is depleted. A linear relationship is demonstrated between the logarithm of the 14C counts per min per nmol of UDP-N-acetylhexosamine and the logarithm of the concentration of D-glucose in the culture medium over a range of 1 to 20 mumol of D-glucose per ml. The relative rates of appearance of counts from 35SO4(2-) and D-[14]glucosamine in chondroitin 4-SO4 and chondroitin-6-SO4 are used to calculate the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool at each stage in the labeling period. The resulting values are then used to calculate the rates of synthesis of the nonsulfated polymers, namely, chondroitin, hyaluronic acid, and glycoprotein.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

Action of Interferon in Enucleated Cells

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.     

Heat-stable variant of human adenovirus type 5: characterization and use in three-factor crosses.

A variant of human adenovirus type 5 which is heat stable (hs) in vitro has been isolated following three rounds of heat inactivation at 52 C. The variant is genetically stable, both through vegetative viral passage and through recombination into other genetic backgrounds, which suggests that it arises from a single mutation. Three-factor crosses, using this mutant in conjunction with previously described temperature-sensitive mutants, suggest the hs mutation lies near the left-hand end of the genetic map. The mutant has been used to demonstrate the production of reciprocal recombinants in two-factor crosses. The mutational lesion is unknown, but phenotypic mixing occurs in hs times hs-+ infections, which suggests that it lies in a gene specifying a viri on structural protein. Other biological parameters examined have shown no differences from the wild-type hs-+.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.