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The arginine deiminase (L-arginine iminohydrolase, EC 3.5.3.6) from Mycoplasma arthritidis catalyzes the irreversible hydrolysis of arginine and related guanidine derivatives to ammonia and the corresponding ureido analog of the substrate. The kinetic constants Km, kcat, and kcat/Km for the arginine deiminase-catalyzed hydrolysis of L-arginine are equal to 4 micron, 29 s-1, and 7.4 X 10(7) M-1 s-1, respectively, at 25 degrees C and pH 7.2. The enzyme also catalyzes the hydrolysis of L-canavanine, Nalpha-methyl-L-argine, D-arginine, L-homoarginine, L-argininic acid, and guanidine, in order of decreasing second order rate constants (kcat/Km); the second order rate constants for these substrates are 10(-3) to 10(-10) smaller than the rate constant for L-arginine. Twenty-two arginine and guanidine analogs were tested for inhibitory capacity. Only 13 are competitive inhibitors having Ki values in the range 3.2 to 40 mM. These results show that binding of ligands to the enzyme is dominated by electrostatic or hydrogen bonding interactions, or both, of the guanidino and alpha-amino group. Neither citrulline nor ornithine, the end product of arginine degradation in M. arthritidis, is an inhibitor of arginine deiminase from this organism.

The functional roles of IgG and C3b in phagocytosis by human peripheral neutrophils were investigated. Phagocytosis of Staphylococcus aureus in the presence of human serum was severely depressed by heat inactivation of serum at 56 degrees C for 30 min. Experiments with varying particle: leukocyte ratios in the presence of complement-inactivated sera showed that particle-bound C3b can mediate a 10-fold enhancement of the overall phagocytic rate. When sheep erythrocytes were sensitized with either IgG or IgM, only the former were bound to and readily internalized by neutrophils. Erythrocytes sensitized with both IgM and C3b were bound but not internalized. Furthermore, the presence of Fc fragments during incubation of S. aureus or latex beads with neutrophils in the presence of IgG or fresh serum affected a total inhibition of internalization but did not significantly alter adherence. Quantitative data regarding IgG sensitization indicated that bound C3b results in at least a 3-fold decrease in the amount of sensitizing IgG required for 50% maximal phagocytic response by neutrophils. On the basis of the above results, it is argued that particle-bound C3b functions primarily in the adherence phase and that bound IgG serves as a trigger for the internalization phase of phagocytosis.

To determine whether correlation exists between serum concentration of phagocytosis-stimulating factor (PSF) and in vivo phagocytic activity, 2 splenectomized steers were inoculated with Anaplasma marginale, and their serum PSF concentrations were monitored. At the time of the anemic crisis, serum PSF concentrations were elevated five to tenfold.

Cyclic AMP was found in species representative of the three major groups of the archaebacteria. In Methanobacterium thermoautotrophicum starvation for H2 led to a significant increase in cellular cAMP. The findings suggest that the occurrence of cAMP antedates the divergence of the major kingdoms of biology; the observations also imply that cAMP constitutes a very early regulatory molecule.

Incubation of Anaplasma marginale-infected bovine red blood cells in a flow culture system resulted in a decrease of observable marginal bodies. The decrease was twice the degree of hemolysis, indicating that marginal bodies may leave red blood cells without concomitant lysis of the host cell.

Hydrodynamic, chemical, and optical properties of arginine deiminase (EC 3.5.3.6) from Mycoplasma arthritidis are reported for the enzyme isolated from log phase cells. The S020,w and D020,w values of the enzyme are 5.48 S and 5.87 X 10(-7) cm3/s, respectively; the molecular weight is 87,300. Determination of the amino acid composition shows that about 45% of the residues are nonpolar. Another unique feature of the composition is the presence of 36 half-cystine residues. The state of oxidation of the half-cystines appears to be well established as 16 disulfide and 4 sulfhydryl groups. The reaction of 1 sulfhydryl group with 0.3 mM 5,5'-dithiobis(2-nitrobenzoic acid) has a half-life of about 50 min at pH 8. The modified enzyme retains full activity. Two -SH groups are accessible to this reagent in 2 M guanidine hydrochloride, whereas all 4 -SH groups react immediately in 4 M guanidine hydrochloride. Reduction of disulfide bonds with dithiothreitol occurs only to a limited extent in 8 M urea, but is complete in 4 M guanidine hydrochloride. The enzyme loses activity immediately at pH 2.5, but retains full activity upon standing 8 h at pH 9.5 in several buffers. The large number of cystine residues leads to a complex near ultraviolet circular dichroism spectrum with cystine contributions apparently superimposed on contributions from aromatic residues. The far ultraviolet spectrum suggests that the molecule contains about 18% alpha helix. At pH 2.5, beta conformation and disulfide contributions are dominant. Aromatic and alpha bands are reduced considerably at pH 9.5.

Methanobacterium thermoautotrophicum was grown in phosphate-limited chemostat cultures at a dilution rate corresponding to a doubling time of 13.2 h. The cyclic-2,3-diphospho-D-glycerate content of these cells was 8 to 10-fold lower than that of cells grown in batch cultures having a doubling time of 11.5 h. This metabolite accounted for 5% of cell dry weight during batch growth on 2 mM phosphate. In the chemostat the steady-state concentration of phosphate was 4 microM, showing that this methanogen is adapted to highly efficient growth at low phosphate concentrations. Since growth rates were similar in both cultures, the growth rate clearly does not depend on intracellular levels of cyclic-2,3-diphosphoglycerate.