Subcellular Distribution Of Enzyme Research Articles

Subcellular distribution of enzymes involved in ?-glucan utilization in Klebsiella pneumoniae

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of 3H-leucine or with glycerol plus maltose in the presence of 14C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from 3H-labelled and 14C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for 3H- and 14C-radioactivity, a positive deviation from the standard 14C/3H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12. 1. Cytoplasm. Neither amylomaltase nor maltodextrin phosphorylase, well-known soluble enzymes, were identifiable by the double-labelling technique, presumably because these enzymes constitute only a very minor portion of all soluble proteins in the cytoplasm. 2. Periplasm. A prominent protein with a mass of 43000 daltons (43 kD) was found similar to the maltose-binding protein of E. coli K-12 (44 kD). 3. Cytoplasmic membrane. At least 2 proteins with a mass between 40 and 50 kD were detected, minor proteins were seen at ≈ 15 and ≈ 20 kD. One or 2 of the proteins may function as a permease catalyzing the active transport of maltodextrins. 4. Outer membrane. The major protein had a mass of 55 kD, other proteins were found with ≈ 18, ≈48, and ≈140 kD. The major protein may have the same function as the maltodextrin pore protein in E. coli K-12 (55 kD), because K. pneumoniae could grow on 10 μM maltose at practically the same rate as on 10 mM maltose. The 140 kD protein is pullulanase.

Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate.

The properties of the guanylate cyclase systems of outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic guanosine 3',5'-monophosphate (cGMP) levels was observed in incubated slices of these tissues (inner medula greater than outer medulla greater than cortex). This correlated with the proportion of total guanyl cyclase activity in the 100 000 g particulate fraction of each tissue, but was discordant with the relative activities of guanylate cyclase (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble guanylate cyclase of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. Thus, the particulate enzyme could play a key role in regulating cGMP levels inthe intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNo2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNo2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. Moreover, relative responsiveness of the sollble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements may be involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble guanylate cyclase of cortex was clearly more responsive to stimulation by NaN3, Nano2, and phenylhydrazine that was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla cound also be distinguished. Accordingly, regulation and properties of soluble guanylate cyclase, as well as subcellular enzyme distribution, and distinct in the three regions of the kidney.

Protein-carboxyl methyltransferase of the bovine posterior pituitary gland: neurophysin as a potential endogenous substrate.

Abstract— The kinetics of methylation of neurophysin by the enzyme protein‐carboxyl methyltransferase (EC 2.1.1.24) was investigated, together with the subcellular distribution of enzyme and endogenous substrate in the bovine posterior pituitary gland. Using a preparation of the methyltransferase, purified from acetone‐dried powders of bovine posterior pituitary gland, bovine neurophysin was found to be a better substrate than those proteins usually employed, neurophysin having a lower apparent Km and higher Vmax than the other substrates. Arginine‐vasopressin did not inhibit the methylation of neurophysin, supporting the hypothesis that methylation and hormone‐binding sites are different. Vasopressin, however, increased the rate of methylation of neurophysin I by the methyltransferase. but was without effect on the rates of methylation of the other components of bovine neurophysin. After subcellular fractionation of fresh bovine posterior pituitary glands, some 77% of the recovered methyltransferase activity was found in the high‐speed supernatant fraction, being distributed throughout all the fractions similarly to lactate dehydrogenase. Neurophysin is therefore unlikely to become a substrate until released from the neurosecretory granules during stimulation of the gland. Some evidence is presented for the presence of an endogenous substrate other than neurophysin, in the high‐speed supernatant fraction.

Lysosomal enzymes and aging in vitro: Subcellular enzyme distribution and effect of hydrocortisone on cell life-span

The acid phosphatase and β glucuronidase activities of four subcellular fractions (nuclear, mitochondrial-lysosomal, microsomal, supernatant) of WI-38 cells were compared during in vitro aging. All of the fractions showed an age-associated increase in activity. The increase in the lysosomal fraction was sufficient to account for the increase in the whole homogenate. The supernatant fraction showed a consistent and pronounced increase suggesting a decrease in latency. Hydrocortisone stabilized the lysosomes to some extent. However the presence of hydrocortisone (5 μg/ml) in the growth medium consistently extended the life-span of the culture 20–30%. The magnitude of the extension seemed to be directly proportional to the amount of time the cultures were exposed to the added hormone.

Relationship between the rate of axoplasmic transport and subcellular distribution of enzymes involved in the synthesis of norepinephrine.

AbstractThe net rate of proximo‐distal transport of tyrosine hydroxylase, dopamine β‐hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β‐hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high‐speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non‐particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β‐hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β‐hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β‐hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non‐particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high‐speed supernatant fluid.

Properties and subcellular distribution of enzymes required for acetoacetate biosynthesis in chicken liver

1. 1. Evidence is presented which indicates that the rate-limiting enzyme of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) pathway of acetoacetate synthesis in chicken liver is HMG-CoA synthase as it is in other species. 2. 2. Maximum rates of acetoacetate synthesis in the catalytic assay system, in which acetyl-CoA is generated from acetyl phosphate and coenzyme A in the presence of purified phosphotransacetylase, was observed when the reaction mixture contained 1 mM coenzyme A and was supplemented with equimolar amounts of both Mg 2+ and Mn 2+. 3. 3. On the basis of studies on the subcellular distribution of HMG-CoA synthase and the other enzymes involved, it is estimated that acetoacetate synthesis capacity is about evenly divided between mitochondrial and cytoplasmic fractions.

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.

Cholesterol ester metabolism in the brain: Properties and subcellular distribution of cholesterol-esterifying enzymes and cholesterol ester hydrolases in adult rat brain

1. 1. The properties and subcellular distribution of the enzymes directly related to the formation and degradation of cholesterol esters were investigated in the young adult rat brain. They were a cholesterol-esterifying enzyme, lecithin-cholesterol acyltransferase, and two distinct cholesterol ester hydrolases. 2. 2. The cholesterol-esterifying enzyme incorporated exogenous free [1- 14C]-oleic acid into cholesterol ester. The pH optimum was 5.5–5.6. Exogenous ATP or CoA did not stimulate the activity, and the presence of an excess amount of unlabelled oleyl-CoA did not diminish the incorporation of the radioactive oleic acid. Various bile acids and Tween 20 were inhibitory, particularly at higher concentrations. Under the optimum conditions, the total activity of the brain was approximately 40 μg cholesterol esterified per h per g. Nearly half of the activity was found in the crude mitochondrial fraction. Purified myelin contained no activity. 3. 3. All attempts failed to demonstrate the activity of lecithin-cholesterol acyltranferase in the adult rat brain, using the specific substrate, lecithin labelled at the β-position with [1- 14C]oleic acid. The contribution of this enzyme to the formation of cholesterol ester in normal adult rat brain appears to be, if not nil, negligible. 4. 4. Two distinct cholesterol ester hydrolases were present. One had the pH optimum of 4.2, was activated by deoxycholate, taurocholate, and cholate, and moderately inhibited by Tween 20. Under the optimum condition in the presence of taurocholate, the total activity of this enzyme was approximately 30μg of cholesteryl oleate hydrolyzed per h per g. This enzyme was primarily localized in the crude mitochondrial fraction, and the subfractionation of the crude mitochondrial fraction suggested that this enzyme might be localized in mitochondria, rather than in lysosomes. Purified myelin was devoid of the pH 4.2 cholesterol ester hydrolase. 5. 5. The other cholesterol ester hydrolase had the pH optimum of 6.6. It was strongly inhibited by deoxycholate and activated by taurocholate. Cholate activated the enzyme at a low concentration, but was moderately inhibitory at a higher concentration. At the optimum condition with added taurocholate, the total activity of the brain was about 170 μg cholesteryl oleate hydrolyzed per h per g. The bulk of this enzyme appeared to be localized in microsomes. However, the highly purified myelin fraction was also enriched with this enzyme by more than 3-fold, compared to the starting homogenate, and it retained 20% of the total activity of the pH 6.6 cholesterol ester hydrolase in the homogenate.

Metabolic Studies in a Patient with Hepatic Cytosol Tyrosine Aminotransferase Deficiency

Extract: Metabolic studies on a patient having multiple congenital anomalies and defective hepatic soluble tyrosine aminotransferase activity are presented. The activities of hepatic mitochondrial tyrosine aminotransferase and soluble p-hydroxyphenylpyryvate hydroxylase were normal. When untreated, levels of tyrosine in plasma rose as high as 62 mg/100 ml. He had phenolic aciduri (3.1–4.9 mg/mg creatinine) with p-hydroxyphenylpyruvic acid (0.6-1.8 mg/mg creatinine), p-hydroxyphenylacetic acid (0.7–1.2 mg/mg creatinine), p-hydroxyphenyllactic acid (1.0–1.7 mg/mg creatinine), and N-acetyltyrosine (0.9–1.6 mg/mg creatinine). These metabolites were identified by gas chromatography-mass spectrometry. Excretion of p-tyramine was grossly elevated (17–44 μg/mg creatinine) and was not reduced by orally administered antibiotics. Oral tolerance tests showed rapid conversion of phenylpyruvic acid to phenylalanine and of phenylalanine to tyrosine. Speculation: The results described suggest the need to reexamine subcellular distribution of enzymes both in normal subkects and in patients with inborn errors of metabolism. When methods are developed to fractionate tyrosine aminotransferase, it may become apparent that there are several isozymes under individual genetic control. Accumulation of an intermediary metabolite should not be taken as conclusive evidence of a primary abnormality of its catabolism. The similarity of the findings in the urine of a patient with hepatic soluble tyrosine aminotransferase deficiency and in tyrosyluric neonates suggests that delayed maturation of soluble tyrosine aminotransferase rather than p-hydroxyphenylpyruvate hydroxylase might be present in at least some of these infants.

Changes in subcellular distribution of enzymes during respiratory adaptation of Saccharomyces carlsbergensis.

Research Article| February 01 1970 Changes in subcellular distribution of enzymes during respiratory adaptation of Saccharomyces carlsbergensis D Lloyd; D Lloyd Search for other works by this author on: This Site PubMed Google Scholar L Howells; L Howells Search for other works by this author on: This Site PubMed Google Scholar T G Cartiledge T G Cartiledge Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1970) 116 (4): 25P. https://doi.org/10.1042/bj1160025Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation D Lloyd, L Howells, T G Cartiledge; Changes in subcellular distribution of enzymes during respiratory adaptation of Saccharomyces carlsbergensis. Biochem J 1 February 1970; 116 (4): 25P. doi: https://doi.org/10.1042/bj1160025Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1970 The Biochemical Society1970 Article PDF first page preview Close Modal You do not currently have access to this content.

The subcellular distribution of enzymes in type II glycogenosis and the occurrence of an oligo- α-1,4-glucan glucohydrolase in human tissues

Summary Biochemical analyses of the tissues from a case (B.J.P.) of Type II glycogenosis revealed generalized storage of glycogen of normal structure, the lack of an α-glucosidase active at pH 4.5, and the presence of all other enzymes whose deficiencies have been implicated in other types of glycogenosis. Differential centrifugation of a sucrose homogenate of an unfrozen sample of liver taken by biopsy from this case showed that more than 50% of the activities of glucose-6-phosphatase (EC 3.1.3.9), uridine diphosphoglucose—α-glucan transghico-sylase (EC 2.4.1.11), α-amylase (EC 3.2.1.1), and of an α-glucosidase active at neutral pH were recovered in the 100 000 × g pellet which also contained most of the glycogen of the tissue. Investigation of the substrate specificity of the α-glucosidase at pH 7.1 showed that it was a hitherto unrecognized type of oligo-α-1,4-glucan glucohydrolase. This enzyme formed no glucose from glycogen but acted rapidly on oligosaccharides (maltose and maltotriaose) to yield glucose. The enzyme has been found in biopsy and autopsy liver samples from ten individuals and in nineteen skeletal muscle samples representative of a vaiiety of types of glycogen storage disease.

A cell sap inhibitor of enzymic activity complicating differential centrifugation studies of enzyme location

1. 1. An inhibitor of the activity of a number of particulate enzymes (mitochondrial succinic dehydrogenase and microsomal cholinesterase and glucose-6-phosphatase) is described. This was developed on storage of the cell sap fraction of rabbit liver homogenates at 4°C by what appeared to be an enzymic process. 2. 2. The inhibitory material was heat stable (100°C) and apparently of high molecular weight, as it was not ultrafiltrable. It was non-protein in nature and apparently partially bound (probably to protein) in the cell sap fraction. 3. 3. Albumin prevented the inhibitory action of cell sap, presumably by binding it and thus rendering it inactive. 4. 4. The inhibitory material became bound to the microsome fraction during the course of the inhibition of microsomal glucose-6-phosphatase activity. The inhibition produced was of two types: ( a) an irreversible progressive inhibition manifested in the absence of substrate; ( b) a competitive reversible inhibition which occurred in the presence of substrate. 5. 5. The inhibitory material appears to have bearing upon techniques and interpretation of differential centrifugation data of subcellular enzyme distribution.