Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.

A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.     

Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria

Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

The pharmacological characteristics, immunochemical identification and study of the location of quisqualate receptors in the rat and human brains]

The kinetics of [3H]-L-glutamate binding to brain synaptic membranes (SM) and to glutamate-binding proteins (GBP) was determined with agonist and monoclonal antibodies (MAbs). It was revealed, that rat and human brain GBP have individual protein components with M(r) from 14 to 92 kDa. Quisqualate inhibited [3H]-L-glutamate binding to solubilized and to purified 68 kDa protein component. MAbs have the most activity, and NMDA was failure. It has been shown that 68 kDa component antigen determinants are similar to those of bovine, frog and rat brain synaptic membranes. Anti-GBP monoclonal antibodies blocked functional non-NMDA receptors in isolated frog spinal cord. Immunocytochemistry was done on rat and human brain sections. Distribution of quisqualate receptors was determined with light and electron microscopy. Some properties of vertebrate CNS non-NMDA receptors are discussed.     

Kinetics of a Novel Isoform of Phosphate Activated Glutaminase (PAG) in SH-SY5Y Neuroblastoma Cells

We have recently found that the neuroblastoma cell line SH-SY5Y expresses a novel form of phosphate activated glutaminase (PAG) which deamidates glutamine to glutamate and ammonia at high rates. Glutamate production is enhanced during the exponential phase of growth, and decreases when cell proliferation stops. Neuroblastoma PAG exists in a soluble and membrane associated form, and both the phosphate and the glutamine kinetics, as well as the effects of ammonia and glutamate are different from those of the known forms of PAG. Neuroblastoma PAG is mitochondrial, and our immunoblotting analyses of isolated mitochondria shows that our C-terminal antibody reacts with a protein of 65 kDa, while our N-terminal antibody primarily labels a protein of 58 kDa and to a minor degree one of 65 kDa. This strongly suggests that neuroblastoma cells mainly contain an active isoform of PAG lacking the C-terminal end, probably the GAC form.     

Biochemical characterization of the microsporidian Nosema bombycis spore proteins

Spore proteins of the microsporidian Nosema bombycis, from the silkworm Bombyx mori, were analysed by SDS–polyacrylamide gel electrophoresis. The protein profile of partially solubilized spores showed three major peptide bands of molecular weight 68, 94 and 100 kDa. On complete solubilization, it showed peptide bands ranging from 17 to 68 kDa. Attempts to purify the 17 kDa infection-specific protein showed aggregation of this protein to higher molecular size proteins. Partial peptide analysis of the different peptides exhibited similar patterns suggesting the probablity of processing during the infective cycle. Reconstitution assay showed the reversible nature of this processing. N-terminal sequencing showed homology to heat shock proteins. The low molecular weight 17 kDa protein also showed very high protease activity.     

A calmodulin endoproteinase from mitochondrial membranes

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.     

Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.     

Evidence that the enoyl-CoA hydratase bifunctional protein of mouse liver peroxisomes is identical with the 70,000 dalton peroxisomal membrane protein

Peroxisomal enoyl-CoA hydratase was purified from livers of mice treated with di-(2-ethylhexyl)phthalate and its properties compared with those of the 70 kDa protein present in the membranes prepared by carbonate extraction of peroxisomes. The two proteins had identical subunit molecular masses, of about 70,000 daltons. Limited proteolysis of these proteins using the V8 proteinase of S. aureus yielded identical peptide maps, with these peptides crossreacting with antiserum raised against the 70 kDa membrane protein. These data are consistent with the proposal that the peroxisomal 70 kDa membrane protein and the peroxisomal enoyl-CoA hydratase are the same protein.     

The TRH-related peptide pyroglutamyglutamylprolinamide is present in human semen

We have recently identified a novel peptide in the rabbit prostate complex which cross-reacts with an antibody to thyrotrophin-releasing hormone (TRH) and has the structure pGlu-Glu-ProNH2. In the present study, high concentrations of a TRH-related tripeptide and also a polypeptide (10-12 kDa) containing a TRH-immunoreactive peptide at its C-terminus were detected in human semen. The low molecular mass TRH-like peptide and the immunoreactive fragment from the polypeptide were isolated from human semen and shown to have identical structures. Amino acid analysis suggested compositions Glx2, Pro1, and after mild acid hydrolysis, the same sequence, Glu-Glu-Pro, was established for the two peptides. Fast atom bombardment (FAB) mass spectrometry yielded a pseudomolecular ion (M + H)+ of 355.38 which was identical to that of the synthetic peptide pGlu-Glu-ProNH2. The data demonstrate that human semen contains the TRH-like peptide pyroglutamylglutamylprolinamide and also a polypeptide terminating in the sequence Gln-Glu-ProNH2.     

Analysis of the major integral membrane proteins of peroxisomes from mouse liver

Two major proteins with subunit molecular masses of 68 and 70 kDa were isolated from the integral membrane protein fraction of peroxisomes purified from mouse liver. The two proteins were shown to be distinct proteins by two criteria: first, immunoblot analysis demonstrated that antisera against the 68 kDa protein did not cross-react with the 70 kDa protein, and vice versa; and second, the partial peptide maps resulting from proteinase digestion of the proteins were different. Immunoblot analyses to test the specificities of the antisera demonstrated that only the expected molecular mass species in purified peroxisomes, and in membranes prepared from these organelles, were recognized; there was no identification of proteins from purified mitochondrial or microsomal fractions. The concentrations of both of these proteins were increased in livers of mice treated with clofibrate, a hypolipidemic drug and peroxisome proliferator, with the effect being greater for the 70 kDa component. The localization of the 68 kDa protein was shown to be completely integral to the peroxisome membrane. Although some 70 kDa protein was integral to the membrane, a significant proportion was released from the membrane by some procedures believed to detach peripheral proteins. The 70 kDa protein was also particularly susceptible to degradation during isolation - in particular, addition of EDTA to media used for isolation of peroxisomes resulted in membranes in which this protein was degraded to smaller immunoreactive fragments. These data have been discussed in relation to the significant clarification which they have provided of the status and characteristics of the major protein components of peroxisomal membranes.     

Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.     

Structural comparison of the 68 kDa laminin-binding protein and 5′-nucleotidase from chicken muscular sources: Evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5′-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5′-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5′-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may possess a few common peptides of yet unknown sequence and length.     

Macluralisin — a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid.

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.Abbreviations DFP diisopropylfluorophosphate - PMSF phenylmethylsulfonylfluoride - Glp pyroglutamyl - NHC₆H₄NO₂ p-nitroanilide This work was supported in part by a grant from the Russian Foundation for Basic Research.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interdomain cleavage of plasma fibronectin by zinc-metalloproteinase from Serratia marcescens

Limited proteolysis of porcine plasma fibronectin by the 56 kDa proteinase (56K proteinase) (EC 3.4.24.4) from Serratia marcescens released six polypeptides: a 27 kDa peptide, the heparin-binding domain which comprises the NH2-terminal end; a 50 kDa peptide, a mid-molecule that mediates binding to gelatin or collagen; a 160 kDa peptide, that contained the heparin-binding domain with cell-spreading activity; and a 140 and a 20 kDa peptide which released from the 160 kDa peptide. Each fragment was purified and characterized by its chemical and biological properties, and it was found that they were respectively different domains. Both the 160 and the 140 kDa peptide contained one cysteine per mole of peptide. The 160 kDa peptides were connected by a 6 kDa peptide, which was present at the COOH-terminal end of the molecule and was biologically inactive. Only 6 kDa peptide contained a disulfide bond and produced 3 kDa peptide after reduction, whereas other fragments did not change with or without reduction on SDS-polyacrylamide gel electrophoresis. NH2-terminal sequence analyses of the released peptides showed that the 56K proteinase cleaved the fibronectin between the Arg-Thr (located at two different sites), Leu-Ser and Gln-Glu bonds. Out of 118 Arg residues, there are nine sequences containing Arg-Thr, and two of them near or at an interdomain location (at Arg 259 and 2239) were cleaved. Out of 124 Leu residues, there are 11 Leu-Ser sequences and only one, at 687, was cleaved. The above fragments with functional domain activity could be aligned according to the previously reported amino-acid sequence of human or bovine plasma fibronectin. The treatment of fibroblast cells by the 56K proteinase resulted in loss of morphological integrity and extracellular matrix.     

Isolation and Characterization of Two Fatty Acid Binding Proteins from Mouse Brain

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.02 and 0.5 µ M , respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.01 and 0.7 µ M , respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. [³H]Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1 and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.