Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Tissue distribution of calcium-activated neutral proteinases in rat

A simple separation method involving a minimum number of essential steps was established to separate the two kinds of calcium-activated neutral proteinase (CANP) activity from each other and from endogenous CANP inhibitor activity. Determination of CANP activity in rat tissues using this method revealed a ubiquitous distribution of two CANPs. The level of CANP activity, however, differs between tissues. Low-calcium-requiring CANP (microCANP) is plentiful in spleen and kidney, while high-calcium-requiring CANP (mCANP) is plentiful in lung and brain. In all of the tissues examined, mCANP activity predominates over microCANP activity.     

Subcellular distribution of aromatic amino acid transaminases in rat brain

Abstract— The subcellular distributions of tyrosine transaminase, DOPA transaminase, tryptophan transaminase and 5-hydroxytryptophan (5-HTP) transaminase were studied in rat brain.

• 1 For all of these transaminases 60-81 per cent of the total activities were found in the crude mitochondrial fraction. Tyrosine transaminase was the most active enzyme.

• 2 Tyrosine transaminase and DOPA transaminase had very similar distributions in all fractions, but the distribution of tryptophan transaminase and 5-HTP transaminase differed in the microsomal (Mic) and synaptic vesicle (M₂) fractions. Only 5-HTP transaminase was highly concentrated in the M₂ fraction.

• 3 DOPA transaminase was inhibited by dopamine and 5-HT, but these compounds had no effect on 5-HTP transaminase. Both enzymes were completely inhibited by m-hydroxybenzoyloxyamine.

     

Subcellular distribution of N-ethylmaleimide-sensitive and -insensitive phosphatidic acid phosphohydrolase in rat brain

The dephosphorylation of phosphatidic acid by phosphatidic acid phosphohydrolase (PAP) is important in both cell-signalling and in glycerolipid metabolism. However, these roles are apparently performed by two different enzymes, which can be distiuguisged by their sensitivity in vitro to N-ethylmaleimide (NEM) Both of these enzymes are present in rat brain as well as a wide range of other rat tissues. However, the quantity and specific activity of each enzyme varies considerably between different tissues, as does the ratio of the two enzymes in each tissue. Tissues rich in glycerolipids are abundant in NEM-sensitive PAP, whereas there is no obvious pattern to the distribution of the NEM-insensitive enzyme in the different tissues tested. Studies on brain cortex, which is relatively rich in both forms of PAP, indicate that the NEM-insensitive PAP is located in the synaptosomes, and the NEM-sensitive enzyme present in the cytosol and microsomes. The NEM-sensitive PAP can also be translocated from the cytosol to the microsomes by oleate. When assayed against a range of phosphatidic acids, NEM-sensitive PAP showed a preference for phosphatidic acids with short acyl chains and for those containing arachidonate, whereas NEM-insensitive PAP had a preference for short and unsaturated acyl chains. The two isozymes also had different activity profiles against these substrates suggesting that they are in fact different enzymes. The implications for these results on the putative roles of the two forms of PAP are discussed.     

Subcellular distribution of lysophosphatidylinositol and lysophosphatidylcholine acyltransferases in rat brain.

Lysophosphatidylinositol acyltransferase utilizing arachidonoyl CoA and lysophosphatidylcholine acyltransferase utilizing linoleoyl CoA were measured in subcellular fractions of rat brain. In general, the distribution of activities paralleled that of NADPH--cytochrome c reductase. It is concluded that the endoplasmic reticulum is the major site of these acyltransferase activities in rat brain.     

Subcellular distribution of acid and neutral alpha-glucosidases in normal, acid maltase deficient, and myophosphorylase deficient human skeletal muscle

The subcellular distribution of acid (pH 4.0) and neutral (pH 6.5) α-glucosidases was investigated in biopsy specimens of human skeletal muscle obtained from six normal subjects, four adult cases of acid maltase deficiency, and a case of myophosphorylase deficiency. The highest relative specific activity of acid glucosidase, as well as of other acid hydrolases, was observed in the light mitochondrial fraction. Relatively high acid phosphatase activity was also found in the microsomal fraction. In all muscles the highest relative specific activity of neutral glucosidase was in the microsomal fraction. In acid glucosidase deficient muscle no neutral glucosidase could be detected in the light and heavy mitochondrial fractions but in normal and myophosphorylase deficient muscle neutral glucosidase activity was also detectable in these fractions. The final supernatant of all muscles contained neutral glucoamylase activity. The relevance of these data to the pathogenesis of the different forms of type II glycogenosis is considered.     

Human placental aldehyde dehydrogenase. Subcellular distribution and properties

Freshly obtained human term placentae were subjected to subcellular fractionation to study the localization of NAD-dependent aldehyde dehydrogenases. Optimal conditions for the cross-contamination-free subcellular fractionation were standardized as judged by the presence or the absence of appropriate marker enzymes. Two distinct isozymes, aldehyde dehydrogenase I and II, were detected in placental extracts after isoelectric focusing on polyacrylamide gels. Based on a placental wet weight, about 80% of the total aldehyde dehydrogenase activity was found in the cytosolic acid and about 10% in the mitochondrial fraction. The soluble fraction (cytosol) contained predominantly aldehyde dehydrogenase II which has a relatively high Km (9 mmol/l) for acetaldehyde and is strongly inhibited by disulfiram. The results indicate that cytosol is the main site for acetaldehyde oxidation, but the enzyme activity is too slow to prevent the placental passage of normal concentrations of blood acetaldehyde (less than 1 mumol/l) produced by maternal ethanol metabolism.     

Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain.

—Certain of the sulphur containing amino acids have been associated with synaptic transmission in the central nervous system. The enzymes involved in the synthesis of these putative neurotransmitter or modulator compounds have a different subcellular distribution in rat brain from those enzymes that catalyse the synthesis of other compounds in this pathway. Methionine adenosyltransferase and 5-methyltetrahydrofolate-homocysteine methyltransferase catalyse reactions that maintain the methylation functions of the pathway and are found in soluble fractions. Cystathionine β-synthase, cystathionase, cysteine dioxygenase and cysteine sulphinic acid decarboxylase catalyse the synthesis of those sulphur-containing amino acids implicated in neurotransmitter functions and these enzymes have both paniculate and soluble components. Serine hydroxymethyltransferase, which also has a particulate fraction in brain, is responsible for the synthesis of the neurotransmitter glycine, in addition to its role in the methionine-related metabolism of folate.     

Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.     

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.