The cardiotropic, hypometabolic and hypothermic activity of peptide fractions from the tissues of hibernating cold-adapted animals

From tissues of hibernating and active long-tailed ground squirrels and from the brain of cold-adapted Yakut horses, low molecular peptide fractions were obtained which, after injection to albino mice, decreased oxygen consumption and rectal temperature in them. The same fractions exhibited negative chrono- and inotropic effects on isolated hearts of ectothermic and endothermic animals. Fractions from the brain of ground squirrels and the brain of horse exhibited similar pattern of the activity. The activity of fractions was subjected to seasonal changes and depended on the degree of their purification. Provisional intracellular microelectrophysiological analysis of the effect of these fractions on the frequency and strength of contractions in isolated heart was made.     

Purification and identification of endogenous anti-opioid substances from bovine brain

Two fractions with high potency for reversing the inhibitory effect of Met-enkephalin on the electrically induced contractility of guinea-pig ileum have been purified from bovine brain extract. Unexpectedly, one isolated peptide was identified as [Val5]-angiotensin I and the other fraction was [Val5]-angiotensin II, as judged by chromatographic comparisons on HPLC and amino acid analysis. Since angiotensins did not affect opioid binding to brain membrane, we consider that angiotensins may act as physiological antagonists to the opioid system in the brain, as well as in the guinea-pig ileum.     

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain.

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.     

The Brain 68-Kilodalton Microtubule-Associated Protein Is a Cognate Form of the 70-Kilodalton Mammalian Heat-Shock Protein and Is Present as a Specific Isoform in Synaptosomal Membranes

The relationship between the 68-kilodalton microtubule-associated protein (68KMAP) and the major heat-induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat-shock cognate gene. Two-dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (alpha 68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (beta 68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that alpha- and beta 68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.     

Polyamines Differentially Inhibit Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

The effects of the naturally occurring polyamines spermine and spermidine on phosphorylation promoted by cyclic AMP (cAMP)-dependent protein kinase (PK) (cAMP-PK; EC 2.7.1.37) were studied using the brain of the tobacco hornworm, Manduca sexta. Four particulate-associated peptides (280, 34, 21, and 19 kilodaltons) in day 1 pupal brains are endogenous substrates for a particulate type II cAMP-PK. These phosphoproteins are present in brain synaptosomal, as well as microsomal, particulate fractions but are not present in the cytosol. They are distributed throughout the CNS and PNS and are present in several nonneuronal tissues as well. Phosphorylation of these proteins via cAMP-PK was inhibited markedly by micromolar concentrations of spermine and spermidine. Other particulate-associated peptides phosphorylated via a Ca2+/calmodulin-PK or Ca2+ and cAMP-independent PKs were unaffected by polyamines, whereas the phosphorylation of a 260-kilodalton peptide was markedly enhanced. Spermine did not exert its inhibitory effect indirectly by enhancement of cAMP or ATP hydrolysis or via proteolysis, but its action appears to involve a substrate-directed inhibition of cAMP-PK-promoted phosphorylation as well as enhanced dephosphorylation. Although addition of spermine resulted in marked ribosome aggregation in synaptosomal and microsomal particulate fractions, this phenomenon was not involved in the inhibition of cAMP-PK-promoted phosphorylation.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides

Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Brain processing of hemorphin-7 peptides in various subcellular fractions from rats

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.     

Peptide hydrolase activity in brain tissues during the early stages of post-hypothermia period

The acid peptidohydrolase activity in the homogenate, dissoluble and mitochondrial-lysosomal fractions of brain tissues of rats who have endured deep hypothermia was determined after their "active" warming for an hour and on the 1st, 2nd, 3d and 7th days after their self-warming. The "active" warming of rats who have endured deep hypothermia (19-20 degrees C) brings about the restoration of the acid peptidohydrolase activity in the subcellular brain tissue fractions. After self-warming the examined enzyme activity restores 7 days later. In the dynamics of the posthypothermic period a change in the acid peptide hydrolase distribution in fractions occurs on the 2nd-3d days.     

Melanocyte-Stimulating Hormone Release-Inhibiting Factor-1 (MIF-1) Can Be Formed from Tyr-MIF-1 in Brain Mitochondria but Not in Brain Homogenate

Abstract: Two samples of the peptide tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH₂) were tritiated on different amino acids (Tyr or Pro) and incubated together at 37°C with fractions of rat brain. The amount of intact tetrapeptide remaining was determined by HPLC. By 3 min, most of the Tyr-MIF-1 was degraded. Because similar amounts of [³H]Pro and [³H]Tyr appeared after incubation of the Tyr-MIF-1 peptides in brain homogenate, even as early as 30 s, examination of only this crude preparation would misleadingly indicate that Tyr-MIF-1 is not a precursor of melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1; Pro-Leu-Gly-NH₂) in brain tissue. However, incubation of the mitochondrial fractions of brain under the same conditions resulted in more than three times as much [³H]Tyr being formed as [³H]Pro, with accompanying accumulation of MIF-1. Addition of excess MIF-1 to the mitochondrial fraction completely suppressed the formation of MIF-1 and more than doubled the amount of Tyr-MIF-1 remaining intact. When Tyr-MIF-1 tritiated only on the Tyr was added to the mitochondrial fraction, the main peaks of radioactivity appeared only at the positions of Tyr and Tyr-MIF-1, not at the position of Tyr-Pro. The results indicate that Tyr-MIF-1 can serve as a precursor of MIF-1 in brain mitochondria, an effect not evident when crude brain homogenate is used.     

Changes in hexokinase isoenzymes in regions of rat brain during thyroid deficiency

In this work, activities of hexokinase isoenzymes Type I and Type II were measured in the soluble and particulate fractions from the brain regions (cerebral hemispheres (cerebrum), cerebellum and brain stem) of the thyroidectomized adult rats as well as of the thyroidectomized rats administered with triiodothyronine. Thyroidectomy generally decreased the hexokinase activity associated with particulate and soluble fractions. Hexokinase Type II isoenzyme was more affected than the Type I isoenzyme. Administration of triiodothyronine to the hypothyroid rats abolished the effect of thyroidectomy. Adult brain enzymes have been generally considered not be affected by thyroid hormones. The data obtained in this work are suggestive of an effect of thyroid hormones on hexokinase in the adult brain. Since the effects of thyroidectomy on the energy metabolism of the heart tissue are well known, the heart tissue was also studied for comparison.     

An inhibitor of opiate receptor binding from human erythrocytes identified as a glutathione-copper complex.

A “peptide-like” inhibitor of opiate receptor binding and of N-methyltransferase previously purified by us from rabbit brain was also found in human red blood cells. Boiled extracts of erythrocytes were fractionated on Sephadex followed by chromatography of the active fractions on silica gel layers. Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate from the origin coincided with the active fractions. The blue substance was identified as copper and the ninhydrin-positive material was identified as oxidized glutathione. While glutathione $\text{per se}$ has no effect, copper and other transition metals are potent inhibitors of opiate receptor binding. A mixture of glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter. Several other laboratories have extracted, from various tissues and body fluids, “opiate-like peptides” which are distinct from the β-LPH derived endorphins. In view of our findings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating factor in some of these studies.     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: Purified rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca²⁺/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca²⁺-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC₅₀=0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca²⁺/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.     

Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe

Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.     

Brain endopeptidase generates enkephalin from striatal precursors

An enzyme capable of converting putative opioid peptide intermediates to free enkephalin has been purified 300-fold from washed rat brain membranes. The action of this enzyme, an enkephalin-generating endopeptidase (EGE), was compared with the action of carboxypeptidase B after trypsin treatment on enkephalin precursor peptides present in rat striata. After Sephadex G-100 gel filtration of striatal material, fractions were radioimmunoassayed for enkephalin content using an antiserum specific for the carboxyl terminal of enkephalin. Additionally, aliquots of the column fractions were treated with either trypsin and carboxypeptidase B, trypsin and EGE, or EGE alone. The peak of enkephalin immunoreactivity increased with the enzymes' treatment indicating the conversion of the low molecular weight proenkephalin precursor peptides to enkephalin. Trypsin and EGE generated almost as much enkephalin as trypsin and carboxypeptidase B in the conditions of the experiment. Thus EGE is capable of processing precursors to enkephalin after the action of trypsin-like enzyme(s) in the brain. The gel filtration fractions containing enkephalin and its low molecular weight precursors were pooled and one-half treated with EGE. The contents were analyzed by HPLC and the increase in immunoreactivity co-eluted with enkephalin and Leu-enkephalin. Small peptides found to be the most potent competitive inhibitors of this enzyme are Met-Arg-Phe-Ala, and Met-Arg-Phe.     

Action of the "delta-sleep peptide" on monoamine oxidase and acetylcholinesterase activity in subcellular fractions from different rabbit brain formations in vivo

Administration of delta-sleep-inducing peptide (DSIP) in vivo in a dose of 30 microgram/kg bw brings about MAO-A (substrate-serotonin) activation in synaptosome subfractions and cellular mitochondria from the brain structures (motor cortex, nucleus caudatus, thalamus). Activity of MAO-B (substrate-p-nitrophenylethylamine) and acetylcholinesterase was inhibited negligibly and specifically in subcellular fractions of the test brain structures. The results suggest that DSIP effects the regulatory or modulation function in the synapse. As one of the elements of sleep mechanisms this peptide induces a number of processes, particularly in serotonin metabolism.     

Effect of brain acid extract products in inbred mice on 3H-diazepam reception

The influence of brain acid extract products, isolated by high-performance liquid chromatography on H3-diazepam binding was investigated in synaptosomal membranes of C57BL/6 and BALB/c mice. Fractions with stimulatory and inhibitory activity were isolated. Quantitative and qualitative differences in the effects and structure of ACTH-immunoreactive peptide fractions under study were established.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Metabolic features of the adaptive effect of delta-sleep inducing peptide and piracetam under hyperoxic conditions.

Adaptive effects of delta-sleep inducing peptide (DSIP, 12 microgram/100 g body weight, single intraperitoneal injection) and piracetam (3 mg/100 g body weight, daily intraperitoneal injection for 3 days) are manifested via differential changes in neurotransmitter amino acids (GABA, glutamate, aspartate), modulation of transport ATPase activity, and decreased accumulation of lipid peroxidation products (conjugated dienes, malonic dialdehyde, Schiff bases) in various fractions of neuronal membranes (myelin, synaptic and mitochondrial membranes) in the sensomotor cortex of rat brain. Under hyperbaric oxygenation (0.3 MPa for 2 h), the combination of DSIP and piracetam enhanced the protective effect of each compound.