Search

Methods for the partial purification of the phosphoprotein phosphatase of the frog egg are described. The enzyme is soluble in distilled water, is heat stable, and is not inactivated by 1 m acetic acid or 0.1 n Na2CO3. The pH optimum (3.0) is unusually low. The enzyme is inhibited competitively by phosphate and molybdate. The enzyme has no activity on the internal pyrophosphates of dinucleotides and similar compounds, on phosphoramidates, or on aliphatic phosphate esters. The enzyme is active on a long series of acyl phosphates: organic and inorganic polyphosphates, both long and short chains and small rings; aromatic phosphates; phosphoenolpyruvate; acetyl phosphate; and carbamyl phosphate. It is less active on phosphoproteins. Evidence is presented which suggests that these activities are all due to one enzyme. The enzyme most closely resembles the phosphoprotein phosphatase of spleen but differs from the latter in several critical respects. The implications of these findings are examined. It is suggested that the enzyme is better classified as a nonspecific phosphoanhydride hydrolase than as a phosphoprotein phosphatase.

Evidence, consisting of analysis of growth requirements and assay of thiamine and precursors accumulated by various strains of Neurospora, strongly suggests that there are two alternative pathways of thiamine biosynthesis in this organism, but that the predominant pathway involves the condensation of pyrimidine with a precursor of thiazole to form a thiamine-like intermediate which is secondarily changed to B1. The reactions are summarized in Eq. (1) (Introduction). These reactions explain the anomalous behavior of those strains which require exogenous pyrimidine and thiazole for growth but synthesize as much of these compounds as does the wild type. The implications of these findings are briefly discussed.

The volume changes of rat liver mitochondria exposed to hypotonic solutions or to solutions of penetrating non-electrolytes were studied by direct estimation of mitochondrial volume from photomicrographs and by changes in optical density of mitochondrial suspensions. The data permit the following conclusions about the osmotic behavior and permeability of these particles. 1. 1. Mitochondria undergo reversible volume changes in anisotonic solutions. 2. 2. Minor secondary changes of undetermined origin also occur. 3. 3. Mitochondria obey osmotic (Boyle-van't Hoff) law. 4. 4. They contain a substantial amount (40–50%) of material which does not participate in volume changes. 5. 5. There appears to be little leakage of osmotically active solute from mitochondria during short exposure to hypotonic solutions. 6. 6. A simple, empirical relation between the optical density of a mitochondrial suspension and mitochondrial volume has been established. 7. 7. Non-electrolytes permeate the mitochondria and thereby induce reversible osmotic volume changes. 8. 8. The rate of penetration is correlated with the olive oil/water partition coefficient of the compound. 9. 9. The permeability constants of the substances studied are quantitatively similar to those found for typical cells. 10. 10. Mitochondria possess a semipermeable membrane which probably contains a high proportion of lipide.

In many cells, a large part of the cytoplasm consists of numerous granules of various types. Undoubtedly they have a real functional significance, but little is yet known of the roles which they may play in cellular processes. This lack of knowledge may be due to the fact that, ordinarily, granules are rather inaccessible to experimental treatment. It was therefore thought worth while to attempt to isolate the granules and study them outside the living cell. This method has the advantage that it enables one to add reagents in known concentrations, and to be certain that they are affecting the granules directly and not secondarily through effects upon the cell itself. Although most granules appear as solid particles, many may actually be minute vacuoles. That this is true of the pigment granules of the Arbacia egg was concluded by Chambers (1935) who found that the pigment escapes when these granules are punctured with a micro-needle. In preliminary studies, I was able to confirm this observation and to provide additional evidence that the pigment granules are actually vacuoles. The most cogent part of this evidence is the fact that in the presence of calcium, magnesium, and strontium ions, the pigment granules (and some colorless granules) coalesce with each other to form large fluid vacuoles from which the pigment soon diffuses, revealing small particles inside in active brownian movement. It is difficult to understand how two particles could coalesce in this manner unless they were vacuoles initially. Photomicrographs of this interesting reaction in isotonic CaC12 are shown in Figure 1. Such minute granules or vacuoles should exhibit osmotic activity. That they do was concluded by Lewis and Lewis (1915) who actually observed swelling and shrinking of mitochondria in tissue culture cells placed in anisotonic media, and by Costello (1939) who found that the formed components of the Arbacia egg occupy 41 per cent of the total volume of the egg, while the osmotically

The inhibition by short-chain fatty acids of several metabolic reactions in yeast and cell-free yeast extracts is demonstrated. Some properties of these inhibitions are shown and their similarity to the binding of short-chain fatty acids to proteins is discussed.

In the nutrition of the young, the role of phosphoproteins is, without doubt, an important one. As casein in milk, or as the yellow yolk of many vertebrate and invertebrate eggs, phosphoprotein occurs in substantial quantities. In the frog egg, for example, the yolk platelets occupy almost half the volume of the cell. According to McClendon (5), the yolk of the frog egg consists of phosphoprotein bound to lipide; thus the platelets are a ready source of protein, lipide, and phosphorus. And yet, in spite of the obvious importance of phosphoprotein, little information is available about the manner of its utilization by the developing embryo. It has been assumed that ordinary proteolytic enzymes degrade the protein, and that ordinary phosphomonoesterases split the phosphate from phosphopeptone or from phosphoserine. In the course of an investigation of the distribution of enzymes in the frog egg, a new enzyme (or enzymes) was discovered which is apparently capable of splitting inorganic phosphate from the intact protein molecule. It is the purpose of this paper to present the experiments which led to this conclusion.

All pyridoxineless mutants of Neurospora crassa and Neurospora sitophila grow better if thiamine is also added to the culture medium. On the basis of the evidence which includes ( a ) an analysis of the growth curves; ( b ) the requirement for thiamine at various concentrations of pyridoxine; ( c ) the interrelation of vitamins B 6 , B 1 , and time; and ( d ) assay of the mycelial content of B 6 and B 1 ; it is concluded that B 1 competitively inhibits the destruction of B 6 . B 6 in turn interferes with thiamine biosynthesis, but sufficient endogenous B 1 is present to satisfy normal metabolic requirements, at least so far as growth is concerned. A specific mechanism is suggested which will be further developed in later publications.

It has been shown that pyridoxine inhibits thiamine biosynthesis. This inhibition is overcome efficiently and noncompetitively by thiamine, competitively by pyrimidine, and inefficiently and noncompetitively by thiazole. In view of these findings, the fact that 4-deoxypyridoxine (a known antimetabolite of pyridoxine) can act in an identical fashion, the great structural similarity of these pyridine compounds to pyrimidine, as well as findings published elsewhere (1–3, 6), it is concluded that pyridoxine competitively prevents pyrimidine utilization, probably by acting as a competitive substrate for the enzyme which couples pyrimidine (or pyridoxine) to thiazole.

1. 1. The dependence of the optical density of a mitochondrial suspension on mitochondrial concentration, osmotic pressure, the refractive index of the medium (Fig. 2), and the wavelength of incident light (Fig. 1) have been examined. 2. 2. Empirical equations which describe the observed relation have been deduced and subjected to empirical verification (e.g. eqn. (8)). 3. 3. The measurements necessary for using these equations for calculating mitochondrial volume from photometric data are described. 4. 4. The simplifications resulting from keeping constant the refractive index of experimental and calibrating solutions are pointed out. 5. 5. The application of photometric techniques to a biological problem, the study of permeability, has been discussed. 6. 6. It is pointed out that a number of changes can occur in mitochondria producing changes in optical density which might be spuriously interpreted as volume changes. Techniques for testing whether an observed optical density change is in fact due to osmotic phenomena are described. 7. 7. A rough theoretical basis for the empirical equations is discussed.

In this paper the functional equations governing the X- and Y-functions (introduced in Paper XXII) are solved by an iteration process, starting with the "first approximation" X(1)=1 and Y(1)=e-τ/μ. The iteration of these functions leads to a "second approximation." The solutions in these approximations are then corrected by a semi-empirical method to satisfy, exactly, an integral property of the X- and Y- functions. It appears that, in this fashion, solutions for the X- and Y-functions, with an error probably not exceeding 1 per cent for τ