N-acetyl-L-glutamate synthase of Neurospora crassa. Characteristics, localization, regulation, and genetic control

N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.     

Position dependence of ankle joint dynamics--II. Active mechanics

System identification techniques were used to examine the position dependence of active ankle joint mechanics. Subjects were required to maintain tonic contractions in either the tibialis anterior (TA) or triceps surae (TS) muscles while the ankle was stochastically displaced about different mean angular positions. The dynamic relation between ankle position and torque was determined for each mean position/tonic torque combination; a non-linear minimization technique was used to estimate the three parameters (inertial, viscous and elastic) of a second-order, underdamped system. Whereas the inertial parameter remained essentially invariant across all test conditions, the viscous and elastic (K) parameters became larger as the level of tonic activity increased and as the joint was rotated toward the extremes of the range of motion. The relation between K and torque was linear at all ankle angles. The slope of this relation remained constant at all mean positions during plantarflexor contractions; during dorsiflexor contractions the slope increased as the ankle was rotated from maximum plantarflexion to maximum dorsiflexion. These findings are discussed in terms of: the physiological correlates of ankle mean position, the relative significance of passive and active joint mechanics and contrasts in joint behaviour during active dorsiflexor and plantarflexor contractions.     

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

Spectral and catalytical properties of the sarcoplasmic reticulum Ca-ATPase labeled with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)-carbodiimide

N-Cyclohexyl-N'-(dimethylamino)-carbodiimide (NCD-4) labels three sites in the sarcoplasmic reticulum Ca-ATPase which can be resolved by their spectral properties and by their effects on the catalytical activity of the enzyme. One site is not protectable by Ca2+ ions or by dicyclohexylcarbodiimide and is not essential for catalytical activity. Two Ca2+-protectable sites, whose modification leads to a biphasic inhibition of Ca-ATPase activity, have fluorescence emission maxima at 407 nm and 425 nm. The Ca-ATPase modified by NCD-4 hydrolyses ATP but does not translocate Ca2+ nor does it undergo the conformational changes associated with Ca2+ binding in the native enzyme. High concentrations of Ca2+ induce slow biphasic fluorescence quenching in the Ca-ATPase labeled selectively at the 407-nm site but the signals are largely abolished by modification of the 425-nm site. Both vanadate ions and ATP reverse this Ca2+-induced fluorescence quenching. It is proposed that NCD-4 labels the two high-affinity Ca2+-binding sites of the Sarcoplasmic reticulum Ca-ATPase and that the conformational changes in the modified enzyme may reflect interactions between the two sites.     

Evolution and expression of the transplantation antigen gene family

We have cloned 26 different class I genes that are located in the major histocompatibility complex of the C57BL/10 mouse. Two of the three class I genes found in the H-2 complex encode the H-2Kb and H-2Db antigens; the other 23 class I genes map to the adjacent Tla complex. We have grouped the cosmids containing these genes into three clusters: one cluster links the H-2K and I-A regions, one cluster links the H-2D and Qa-2 regions, and the final cluster maps to the TL region. The class I gene organizations in the Qa-2 and TL regions of the C57BL/10 and BALB/c mice are generally similar, but there are several polymorphic segments. The Qa-2 region of both mice seems to have evolved by the duplication of gene pairs; furthermore, the H-2K region may have been generated by the translocation of a gene pair from the Qa-2 region. We have evidence that several of the genes in the Qa-2 region are expressed.     

Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells

Quantitative studies of MCF-7 cells (derived from human breast adenocarcinoma) and CV-1 cells (from normal African green monkey kidney epithelium), using the permeant cationic compound tetraphenylphosphonium (TPP), in conjunction with fluorescence microscopy using rhodamine 123 (Rh123), indicate that the mitochondrial and plasma membrane potentials affect both uptake and retention of these compounds. Under conditions that depolarize the plasma membrane, uptake and retention of TPP and Rh123, driven only by the mitochondrial membrane potential, is greater in MCF-7 than in CV-1. An ionophore that dissipates the mitochondrial membrane potential of MCF-7 cells causes them to resemble CV-1 cells by decreasing uptake and retention. Hyperpolarizing the mitochondrial membrane of CV-1 increases accumulation and prolongs retention; hyperpolarization of the plasma membrane further heightens this effect, causing the uptake of CV-1 cells to resemble that of MCF-7 cells even more closely. The greater uptake and retention by MCF-7 appears to be a consequence of elevated mitochondrial and plasma membrane potentials. The plasma membrane potential affects mitochondrial retention of TPP and Rh123 and its role in enhancing the effect of a difference in mitochondrial membrane potential is explained.     

Regional Distribution of Calmodulin Activity in Rat Brain

Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin-sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday