Evidence for a new inositol phospholipid in rat brain mitochondria.

Phosphorylation of phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and diacylglycerol (DAG) was studied in rat brain cortex myelin, synaptosomal and mitochondrial fractions, with ATP as phosphate donor and endogenous phospholipids as substrate. All fractions had PI, PIP and DAG phosphorylating activity with their own characteristic subcellular distribution. However, in the mitochondrial fraction an unidentified lipid was phosphorylated, which had a slower Rf value than PIP2 on TLC. After hydrolysis of the polar head group of the lipid and separation on anion exchange columns, it appeared to be a phosphoinositide. The elution profile showed that it was not phosphatidylinositol trisphosphate, or a lyso-compound. The available evidence suggests that the unknown inositol phospholipid in rat brain mitochondria is a phosphatidylinositol 4,5-bisphosphate isomer, although the possibility of it being a glycosyl-phosphoinositide cannot be excluded.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain. Evidence for an activator deficient form.

A cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain was partially purified after solubilization with a non-ionic detergent. Influence of divalent ions, of phosphodiesterase inhibitors and of the activator protein from different sources were tested. Determination of K_m-values shows two enzymes with different values, one at 7.3 μM and the other at 15 mM. Two distinct activity peaks were determined after resolution by isoelectric focusing. It was concluded that this particulate enzyme is regulated in a way opposite to that of the soluble enzyme and is independent from calcium and the activator protein.     

Evidence for a phosphorylated form of calmodulin in chicken brain and muscle.

Phosphocalmodulin (PCaM) was identified after analysis of calmodulin (CaM) preparations by two-dimensional gel electrophoresis by using a modified ampholyte system to resolve very acidic proteins. The analysis of CaM prepared by the conventional procedure based upon its heat resistance and acidity as well as the analysis of whole urea extracts from brain showed that PCaM was a major component in this tissue. PCaM was 1 pH unit more acidic than CaM, and its electrophoretic mobility, unlike CaM, was not changed by either calcium or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. In urea extracts of brain prepared in buffers containing phosphate and sodium fluoride, PCaM was as prominent as CaM; it was partially converted into CaM after elution from the gel and reelectrophoresis. Amino acid analysis of PCaM and CaM purified by two-dimensional gel electrophoresis showed the same composition for the two proteins, including their trimethyllysine content. Incorporation of 32P occurred exclusively into the acidic variant when brain slices were incubated with H332PO4; amino acid analysis showed that the phosphate was bound to serine residues. CaM was found also to be phosphorylated in vitro by a phosphorylase kinase preparation from skeletal muscle.     

The rat kidney acylase 1. Evidence for a new cDNA form and comparisons with the porcine intestinal enzyme

A new cDNA form encoding the rat kidney acylase I was characterized and found to show as much as 93.5% identity in its translated nucleotide sequence and, to a lesser extent, in its 3'-untranslated region with the nucleotide sequence we previously reported in 2000. Comparisons between the amino acid sequences of the two corresponding proteins showed the presence of N-terminal fragments with 88.5% identity and different cysteine profiles. The cDNA nucleotide sequence of the pig intestinal enzyme isolated from a marathon library turned out to be 100% identical to that of the kidney enzyme, but differed from those of the two rat kidney acylase I forms.     

Evidence for a new subclass of calcitonin/ calcitonin gene-related peptide binding site in rat brain

Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [¹²⁵I]-alpha-rat calcitonin gene-related peptide and [¹²⁵I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 × 10⁹ M⁻¹ and 1.2 × 10⁹ M⁻¹ respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [¹²⁵I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 × 10⁹ M⁻¹. However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [¹²⁵I]-salmon calcitonin labelled sites with only 28% inhibition at 10⁻⁶M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [¹²⁵I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [¹²⁵I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe.These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.     

Evidence for a zinc/proton antiporter in rat brain

The data presented in this paper are consistent with the existence of a plasma membrane zinc/proton antiport activity in rat brain. Experiments were performed using purified plasma membrane vesicles isolated from whole rat brain. Incubating vesicles in the presence of various concentrations of 65Zn2+ resulted in a rapid accumulation of 65Zn2+. Hill plot analysis demonstrated a lack of cooperativity in zinc activation of 65Zn2+ uptake. Zinc uptake was inhibited in the presence of 1 mM Ni2+, Cd2+, or CO2+. Calcium (1 mM) was less effective at inhibiting 65Zn2+ uptake and Mg2+ and Mn2+ had no effect. The initial rate of vesicular 65Zn2+ uptake was inhibited by increasing extravesicular H+ concentration. Vesicles preloaded with 65Zn2+ could be induced to release 65Zn2+ by increasing extravesicular H+ or addition of 1 mM nonradioactive Zn2+. Hill plot analysis showed a lack of cooperativity in H+ activation of 65Zn2+ release. Based on the Hill analyses, the stoichiometry of transport may include Zn2+/Zn2+ exchange and Zn2+/H+ antiport, the latter being potentially electrogenic. Zinc/proton antiport may be an important mode of zinc uptake into neurons and contribute to the reuptake of zinc to replenish presynaptic vesicle stores after stimulation.     

Changes in levels of translatable mRNA for neuron-specific enolase and non-neuronal enolase during development of rat brain and liver

Neuron-specific enolase (NSE), and non-neuronal enolase (NNE) which exists in many tissues including liver but is localized in glial cells within the nervous system, were synthesized in the rabbit reticulocyte cell-free translation system programmed with brain mRNAs. The in vitro synthesized NSE and NNE were indistinguishable from the two enzymes purified from rat brains. NSE mRNA activity was found only in brain RNAs, while NNE mRNA activity existed in brain RNAs as well as liver RNAs. In developing brains, the level of translatable NSE mRNA was low at the embryonic stage and at birth, increased rapidly from about 10 days postnatal, and reached the adult level, while that of NNE mRNA was high at the embryonic stage and at birth, followed by a slight decrease then a gradual rise to adult levels. These changes correlated with the developmentally regulated appearance and accumulation pattern of each of the two enzymes. These results suggest that the levels of NSE and NNE are controlled primarily by the level of each of the two translatable mRNAs. In developing livers, only the NNE mRNA activity was detected and its level generally paralleled the changes in the level of NNE.     

Evidence of a precursor form of stratum corneum basic protein in rat epidermis.

The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.     

Evidence for a new activator of rat liver phosphofructokinase

A low molecular weight compound that activates purified rat liver phosphofructokinase has been isolated and partially purified from rat hepatocyte extracts. It can be separated from both fructose bisphosphate and AMP on DEAE-Sephadex. Incubation of rat hepatocytes with glucagon lowers the level of this activator, and this accounts for the inhibition of phosphofructokinase that was observed in hepatocyte extracts (S. Pilkis, et al. (1979) Biochem. Biophys. Res. Commun. $\text{88}$, 960–967). Other characteristics of this activator are described which suggest that it is not any of the known effectors of rat liver phosphofructokinase.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that bacteria can form new cells in airborne particles.

Serratia marcescens incubated for 8 h at 31 degrees C in a chemically defined medium contained in shake flasks was aerosolized into rotating-drum aerosol chambers at 30 degrees C and saturated humidity. Cells furnished tryptone (Difco) and glycerol just before aerosolization increased (in viable numbers and countable cells) almost twofold within 1 to 2 h after becoming airborne, whereas cells not furnished additional tryptone decreased in viable numbers at a faster rate than the number of particles removed by gravitational settling. Limited tests with a Coulter Counter showed that cell volume changes occurred in growing cells that did not occur in the nongrowing population.     

Evidence that bacteria can form new cells in airborne particles.

Serratia marcescens incubated for 8 h at 31 degrees C in a chemically defined medium contained in shake flasks was aerosolized into rotating-drum aerosol chambers at 30 degrees C and saturated humidity. Cells furnished tryptone (Difco) and glycerol just before aerosolization increased (in viable numbers and countable cells) almost twofold within 1 to 2 h after becoming airborne, whereas cells not furnished additional tryptone decreased in viable numbers at a faster rate than the number of particles removed by gravitational settling. Limited tests with a Coulter Counter showed that cell volume changes occurred in growing cells that did not occur in the nongrowing population.     

Evidence for a glycoconjugate form of glutathione S-transferase pI.

The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes.     

Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein

We recently cloned a rat brain agmatinase-like protein (ALP) whose amino acid sequence greatly differs from other agmatinases and exhibits a LIM-like domain close to its carboxyl terminus. The protein was immunohistochemically detected in the hypothalamic region and hippocampal astrocytes and neurons. We now show that truncated species, lacking the LIM-type domain, retains the dimeric structure of the wild-type protein but exhibits a 10-fold increased k_cat, a 3-fold decreased K_m value for agmatine and altered intrinsic tryptophan fluorescent properties. As expected for a LIM protein, zinc was detected only in the wild-type ALP (∼2 Zn²⁺/monomer). Our proposal is that the LIM domain functions as an autoinhibitory entity and that inhibition is reversed by interaction of the domain with some yet undefined brain protein.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.     

Crystallization and preliminary crystallographic data for a tetragonal form of yeast enolase

Yeast enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) has been crystallized by vapor diffusion and equilibrium dialysis of solutions of ammonium sulfate. The crystals with the dimer have 2-fold axial symmetry and appear to be suitable for high-resolution X-ray diffraction analysis. Our potential heavy-atom derivative of the native crystals has been prepared.