In Neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. We wished to know what determines these pool sizes. In the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. In a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. However, this was true only after an intracellular "sink," with a capacity equal to the amount of spermidine found in wild-type cells, had been saturated. We speculate that internal anionic binding sites, detectable in permeabilized cells, sequester virtually all of the spermidine normally found in exponentially growing N. crassa. Further evidence for this view was that in mature, stationary cultures, excess spermidine is excreted. Putrescine is also excreted if its concentration in the cell is abnormally high. The control of pool size by intracellular binding and excretion may be an advantage in this pathway, because feedback inhibition does not prevail, enzyme regulation is by comparison slow, and excessive polyamines are toxic.

Polyamine transport in Neurospora crassa is concentrative and energy dependent in a dilute buffer. The saturable systems governing the uptake of putrescine (Km = 0.6 mM), spermidine (Km = ca. 0.24 mM), and spermine (Km = 0.07 mM) share components, as indicated by mutual inhibition among the polyamines. In addition, nonsaturable components prevail for putrescine and spermidine, particularly the former. Radiolabeled substrates, once in the cell, are released only slowly, even if unlabeled polyamines are included in the incubation medium. Permeabilization of cells with n-butanol leads to partial release of internalized 14C-polyamines, and the remainder is almost wholly exchangeable with added, unlabeled polyamine. Polyamine uptake was inhibited by the polyamines themselves and by a polyamine analog, methylglyoxal bisguanylhydrazone, but only weakly and incompletely by the basic amino acids arginine and ornithine. Uptake of putrescine and spermidine was inhibited by monovalent cations, Ca2+, and certain other components of the growth medium. As a result, uptake from the growth medium was very slow and largely by way of the nonsaturable uptake mechanism.

In bacteria, mammals, and certain plants, the induction of the polyamine synthetic enzyme, ornithine decarboxylase (ODC), and the accumulation of its product, putrescine, follows osmotic manipulations of cells. In at least some of these cases, this response is indispensable for survival. We wished to determine whether the polyamine pathway of Neurospora crassa was regulated in response to hyper- or hypoosmotic conditions. Unlike ODC of most other classes of organisms, the N. crassa enzyme and the accumulation of putrescine appears to be relatively indifferent to these conditions, either during sudden transitions or in steady-state. We conclude that other mechanisms of osmotic adjustment or tolerance have evolved in N. crassa and perhaps other fungi that obviate the need for putrescine accumulation.

We used mutant strains of Neurospora crassa to define the discretionary capacity of this species for excess putrescine. The spe-3 mutant, which accumulates putrescine internally, and the puu-1 mutant, which accumulates toxic levels of putrescine from the medium, both sequestered large excesses of putrescine in vacuoles. Concomitantly in puu-1, inorganic polyphosphate increased modestly and some of the monovalent cation of the vacuole was discharged. These two factors contribute to the increased capacity for polyamines in this fungus. Putrescine, however, can exceed the capacity of vacuoles such that they no longer protect the cytosol from toxic levels of the amine. The puu-1 mutant illustrates the importance of the sequestration of intracellular polyamines, as well as the control of polyamine uptake through the cell membrane.

Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon arginine-starvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.

A new and inexpensive glass-bead blender allows rapid, easy, and controlled cell breakage of Neurospora, with good organellar survival. The yield of mitochondria and vacuoles is comparable to or better than methods involving sand grinding or snail-gut digestion of cell walls. A method for removing cell wall fragments from a crude homogenate is described. Isolation of mitochondria and vacuoles from the crude homogenate with little cross-contamination is accomplished by density-gradient centrifugation in a fixed-angle rotor (Sorvall).

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca(2+)-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-1+ gene. Preliminary evidence suggests that the putrescine carrier is not the Ca(2+)-sensitive, low-affinity K(+)-transport system, but K+ efflux does accompany putrescine uptake.

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.