Vitamin B12-binding proteins of the horseshoe crab Limulus polyphemus.

Vitamin B12-binding proteins were detected in the body fluids and/or tissues of horseshoe crabs (Limulus polyphemus), clams and sponges. Among the biological specimens tested the Limulus plasma was especially rich in free B12-binding proteins. Gel filtration experiments revealed that Limulus plasma contains two classes of B12-binding proteins. One class of proteins, molecular weight in excess of 100,000, bind B12 preferentially with affinity constant of 5 X 10(11)M-1. The second type of proteins, molecular weights around 50,000, bind B12 with specificity approaching that of mammalian intrinsic factors. The binding constant of these proteins for B12 is around 10(11)M-1.

Drug-induced suppression of phospholipid synthesis in HeLa cells by inhibition of choline uptake.

2-(4-Anisidino)-4,6-bis (2-(diethylmethylammonium)ethylamino)-1,3,5-triazine diiodide was found to inhibit specifically the incorporation of [14 C]-choline into cold 5 percent trichloroacetic acid-insoluble materials in HeLa cells without affecting other vital areas of cellular metabolism. Further studies indicated that this drug did not affect any of the intracellular reactions leading to phosphatidylcholine formation; instead, the inhibition of lecithin synthesis was due primarily to the suppression of choline transport through the cytoplasmic membrane. The level of inhibition of the latter process was comparable to that observed for the inhibition of choline incorporation into cold 5 percent trichloroacetic acid-insoluble precipitates in whole cells.

Dependence of diaminopurine utilization on the mutational site of purine auxotrophy in Bacillus subtilis. 1. Nutritional experiments.

An attempt was made to explain the puzzling observation that in bacteria 2,6-diaminopurine can replace guanine for guanineless mutants and for xanthineless mutants (both of which can make adenosine monophosphate de novo) but not for nonexacting purine auxotrophs (which cannot make adenosine monophosphate de novo). The analogue failed to inhibit the growth of nonexacting purineless Bacillus subtilis MB-1356 growing on guanine. In fact, growth was somewhat stimulated. This eliminated a possible solution involving the inhibition of guanosine monophosphate reductase by a diaminopurine derivative. Sparing of guanine by diaminopurine was matched by an even greater sparing of adenine. Addition of a small amount of adenine to MB-1356 failed to allow unrestricted growth on diaminopurine, thus eliminating a possible solution requiring an adenine derivative for the initial deamination of diaminopurine to guanine. The same degree of sparing of adenine by diaminopurine was observed whether both purines were added together or whether the adenine was added 1 hr after diaminopurine. This eliminated the possibility that diaminopurine was wasted by a "dead-end" conversion in the absence of adenine. Consideration of these nutritional data led to the development of two additional explanations, which are examined by tracer methodology in the following paper.

Dependence of diaminopurine utilization on the mutational site of purine auxotrophy in Bacillus subtilis. II. Tracer experiments.

Tracer experiments were carried out in an attempt to explain why guanineless auxotrophs can use diaminopurine as a guanine replacement but nonexacting purine auxotrophs cannot do so. Cell suspensions of the nonexacting purineless Bacillus subtilis MB-1356 incorporated more radioactivity from diaminopurine-2-(14)C into nucleic acid than did guanineless B. subtilis MB-1517. The radioactivity in MB-1356 ribonucleic acid (RNA) was distributed in both adenine and guanine nucleotides, thus eliminating the possibility that the deamination of diaminopurine to guanine occurred predominantly on the level of nucleoside di- or triphosphates. Strain MB-1517 incorporated adenine-8-(14)C into nucleic acids extremely poorly. This correlated with results obtained with cell-free extracts; strain MB-1517 showed much less adenosine monophosphate (AMP) pyrophosphorylase activity than did MB-1356. Likewise, guanineless MB-1517 converted diaminopurine to its nucleotide much more slowly than did the nonexacting purine auxotroph. The results indicated that the lack of growth of nonexacting auxotrophs on diaminopurine alone is due not to an inability to convert the analogue to nucleic acid adenine but to the greater capacity of the nonexacting auxotrophs to convert diaminopurine to its 5'-ribonucleotide. Presumably, this compound, or a coenzyme analogue produced from it, inhibits growth of mutants which cannot make AMP de novo and only when the medium is devoid of adenine.