Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.

     

INTESTINAL TRIGLYCERIDE ABSORPTION IN THE RAT : An Electron Microscopical Study

This report provides information on the morphology of fat absorption in rat intestinal epithelial cells. Three types of experiments were performed: (a) intubation of corn oil into fasted rats, (b) injection of physiological fatty-chyme prepared from fat-fed donor rats into ligated segments of jejunum of fasted animals, and (c) administration of electron-opaque particles in corn oil and markers given concurrently with the fat. These results support the hypothesis that fat is absorbed by selective diffusion of monoglycerides and fatty acids from micelles rather than by pinocytosis of unhydrolized triglycerides. Evidence is presented that the pits between the microvilli, previously believed to function in the transport of fat, are not involved in this process. Instead they appear to contribute their contents to lysosomes in the apical cytoplasm. Arguments are offered that the monoglycerides and fatty acids diffuse from the micelle while the latter is associated with the microvillous membrane of the absorptive cell. These micellar components penetrate the plasma membrane and diffuse into the cytoplasmic matrix where they encounter the SER. Triglyceride synthesis occurs in the SER and results in the deposition of fat droplets within its lumina. The synthesis of triglycerides and their sequestration into the SER establishes an inward diffusion gradient of monoglycerides and fatty acids.     

Comparison of the structure of the immunoglobulins from horse serum

A study of the chemical structure of the horse immunoglobulins IgG and IgA(T) has shown that the amino acid contents of the peptide chains are very similar. These globulins differ most markedly in the products of papain digestion. IgG gives 3.5s products, whereas IgA(T) gives a 5s fraction and smaller components. This difference appears to be associated with the presence of an additional easily reducible disulphide bond in the Fd fragment of the heavy chain. There is two to three times as much carbohydrate in IgA(T) as in IgG. In both, this is in the heavy chain and in IgA(T) more than half is covalently bound to the Fd fragment. The differences in antigenic specificity between horse IgG and IgA(T) appear to be due to structural differences in the Fc fragment.     

Studies on the Endoplasmic Reticulum : IV. Its Form and Distribution during Mitosis in Cells of Onion Root Tip

Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO₄. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO₄-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.     

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.