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Transport of the acidic amino acids by Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The neutral transport system is shown to possess “high affinity”, but low transport capacity for the acidic amino acids. The general transport system is shown to have “low affinity” and high transport capacity for these amino acids. The non-linearity of a Lineweaver-Burk plot of aspartate transport by the wild type has been shown to result from an overlapping of two transport systems for accumulation of this amino acid. Removal, by mutation, of the neutral transport system restores the transport kinetics to a linear function. The combined, and separate, transport activities are shown to be stereospecific, preferring the l-stereoisomeric form over the d-form, and to transport the neutrally charged species of l-aspartate and l-glutamate. The transport activity is dependent on pH, and the V, but not the C12, constant of the general transport system is pH dependent. Two models for amino acid transport are presented as possible mechanisms to explain the observed kinetics. One model involves three genetically distinct transport systems. The second model utilizes a single “allosteric permease” to account for the kinetic data.

Growth of an amino acid auxotroph of Neurospora crassa on oligopeptides is shown to occur by extracellular hydrolysis, with subsequent utilization of monomer amino acid residues, and by transport of peptides. Peptides with a hydrodynamic volume greater than that of trileucine are not transported, and this lack of transport is shown to be due to restriction by the oligopeptide transport system rather than the cell wall.

The tripeptide, glycyl- d , l -leucyl- l -tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora . Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.

A simple purification procedure has been developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: 1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; 2) heat treatment, during which the inducer is digested by the protease; and 3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. These procedures were followed by routine ion exchange chromatography and gel filtration. The preparation was homogeneous, as determined by gel electrophoresis and ultracentrifugal analyses. A molecular weight of approximately 30,500 was determined by amino acid analysis, gel electrophoresis, and sedimentation equilibrium. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45 degrees C, and susceptible to autodigestion. Hydrolysis of the beta chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize L-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3. Transport of L-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.

A double amino acid transport-deficient mutant (Pm − NB) of Neurospora crassa is shown to be altered in the molecular structure of its cell wall or membrane. This alteration was revealed by a high degree of cellular disruption and cell-cell interaction following extraction by a high molar concentration of KCl.