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K Paigen

Publications and source records attributed to K Paigen.

At least 55 records · Page 3Linked to original sources
PubMed1978

Regulation of the rate of beta-galactosidase synthese by the Bgs and Bgt loci in the mouse.

We have developed an assay for the in vivo rate of beta-galactosidase synthesis in mouse tissues to assess the mechanism by which the Bgs and Bgt loci regulate activity levels of this enzyme. Genetically determined differences in liver and kidney beta-galactosidase content reflect equivalent differences in specific rates of enzyme synthesis. We conclude that Bgs and Bgt regulate beta-galactosidase activity by controlling the rate of synthesis of the beta-galactosidase molecule.

Alleles↗
PubMed1978

Segregation of genetic determinants for murine glucuronidase synthesis and loss in CXB recombinant-inbred strains.

A set of recombinant-inbred strains developed from mouse strains BALB/c and C57BL/6 includes two beta-glucuronidase phenotypes that are not seen in either of the progenitor strains. These new recombinant phenotypes indicate that glucuronidase levels are regulated by genes additional to the Gur locus, which is closely linked to the glucuronidase structural gene (Gus) and is known to regulate the rate of glucuronidase synthesis. In this study, induced rates of glucuronidase synthesis were determined for these recombinant-inbred strains, and rate constants for enzyme loss were calculated. The rate of synthesis was found to segregate with the Gus gene in all of the strains, and only the determinants for rate of enzyme loss recombined to give new phenotypes. It was concluded that at least two genes affect the rate of enzyme loss, that these genes are not closely linked to each other or to the Gur-Gus region on chromosome 5, and that no major determinants of glucuronidase synthesis segregate independently of Gur.

Animals↗
PubMed1978

Regulation of mouse major urinary protein production by the Mup-A gene.

A method was developed to quantitate the daily excretion of the three major urinary proteins (mups) to test which parameters of the mup phenotype are controlled by the the Mup-a gene. Electrophoretic separation of the mup proteins, followed by staining and spectrophotometric scanning was used to characterize the phenotypes of various inbred strains. The mup phenotype of a strain proved to have two components: the absolute levels and the relative proportions of the mups present in the urine. Testosterone treatment alters both components of the mup phenotype, increasing mup excretion and aftering their relative proportions. The induced proteins are the same as the basal proteins as judged by electrophoretic mobility, molecular weight, and reactivity with antibody. All strains excrete all three mups when induced. The Mup-a gene appears to be a single, codominantly expressed regulatory locus that controls the induced proportions of the three proteins. However, other genes in addition to Mup-a participate in controlling the basal mup proportions, as well as individual and total mup levels before and after testosterone treatment.

Animals↗
PubMed1978

Coordinacy of lysosomal enzyme excretion in human urine.

Assay conditions have been developed for the determination of urinary beta-glucuronidase, beta-galactosidase, alpha-galactosidase, and beta-hexosaminidase using fluorometric substrates. The assay conditions for beta-glucuronidase overcome interference by both low and high molecular weight inhibitors, a problem that has confused earlier studies of enzyme excretion. The four lysosomal enzymes are excreted corrdinately: although their absolute levels (in units per milligram of creatinine) vary during the day and from one day to the next, the ratio of one enzyme to another remains relatively constant. The lack of correlation betweem plasma and urine enzyme levels, together with the high molecular weights of these enzymes, suggests that the urinary enzymes are not derived by glomerular filtration. The lack of coordinacy with lactate dehydrogenase suggests they are not derived from exfoliated cells. by analogy with experimental animals, they may be derived from lysosomes extruded into the lumen of the proximal tubule by epithelial cells. There is considerable variation among a population of 125 healthy adult subjects for total enzyme excretion. Both total enzyme excretion and coordinacy ratios are log-normally distributed, suggesting that they are the resultants of many factors, each of which has a relative, or proportional, effect on enzyme excretion. About one-half the population variation resides in a process common to the excretion of all four enzymes (possibly the lysosome extrusion pathway), and about one-half resides in factors affecting each enzyme independently.

Adolescent↗
PubMed1977

Questionable relation of aryl hydrocarbon hydroxylase to lung-cancer risk.

To test whether the genetically determined trait, aryl hydrocarbon hydroxylase inducibility, affects susceptibility to lung cancer, we measured this trait in cultured lymphocytes from a normal population, patients with lung cancer and progeny of such patients. We found very low aryl hydrocarbon hydroxylase activity (19 per cent of normal) in about half the patients with lung cancer. Only part of this activity can be accounted for by reduced cell growth and by reduced protein synthesis. In an indirect assessment of inducibility, both 57 progeny and 27 matched controls had a mean inducibility of 2.95 and a similar distribution into low, intermediate and high groups (chi-square = 0.3 P = 0.9). No differences in basal or induced activity were observed. Thus, if patients with lung cancer possess altered aryl hydrocarbon hydroxylase inducibility or activity these characteristics are not transmitted to their progeny.

Adult↗
PubMed1977

Inheritance in mice of the membrane anchor protein egasyn: the Eg locus determines egasyn levels.

Previous studies have suggested that the binding of mouse flucuronidase to endoplasmic reticulum membrane is stabilized by the membrane protein egasyn. Using a radioimmunoassay for egasyn, we have now examined the inheritance of egasyn levels in mice. Mice of the inbred strain C57BL/6J, which have normal levels of microsomal glucuronidase, contained 56 +/- 10 mug egasyn per gram of liver. Mice of the inbred strain YBR, which carry the Eg0 mutation resulting in the absence of microsomal glucuronidase, did not contain detectable levels of egasyn. The F1 progeny of these two strains contained intermediate levels of egasyn, 25 +/- 4 mug egasyn per gram of liver. Progeny from the backcross of these F1 animals to YBR were distributed equally into two discrete phenotypic classes. One class lacked both egasyn and microsomal glucuronidase, while the other class contained 25 +/- 3 mug egasyn per gram of liver and contained normal levels of microsomal glucuronidase. Thus egasyn levels are determined by the Eg locus and show additive inheritance. These results suggest that the Eg gene codes for egasyn and that it is the inability to produce egasyn that results in a deficiency of microsomal glucuronidase in the Eg0 mutant.

Animals↗
PubMed1977

MRNA-directed synthesis of catalytically active mouse beta-glucuronidase in Xenopus oocytes.

Catalytically active mouse beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) is formed when Xenopus oocytes are injected with mouse RNA enriched for poly(A)-containing mRNA sequences. With the RNA from androgen-induced kidneys, the efficiency of translation is comparable to that of endogenous Xenopus messenger, and the fidelity of translation is high. Detection of glucuronidase messenger by formation of a catalytically active product is several orders of magnitude more sensitive than detection by incorporation of isotopically labeled amino acids. As well as providing a sensitive technique for examining the regulation of gene expression, the system makes available an opportunity to study the regulation of post-translational polypeptide processing of a lysosomal enzyme.

Animals↗
PubMed1977

Relationships between levels of membrane-bound glucuronidase and the associated protein egasyn in mouse tissues.

Mouse beta-glucuronidase has a dual intracellular localization, being present in both endoplasmic reticulum and lysosomes of several tissues. Previous studies demonstrated that the protein egasyn is complexed with microsomal but not lysosomal glucuronidase and that a mutant lacking egasyn is deficient in microsomal, but not lysosomal, glucuronidase. By means of a recently developed radioimmunoassay for egasyn, the relationship between microsomal glucuronidase levels and egasyn levels has been examined in various adult tissues, during postnatal development in liver, and after androgen induction of glucuronidase in kidney. The results indicate that the relative availability of egasyn determines the balance between glucuronidase incorporation into membranes and that into lysosomes.

Animals↗
PubMed1977

Linkage of genetic determinants for mouse beta-galactosidase electrophoresis and activity.

An electrophoretic polymorphism for beta-galactosidase has been identified among common inbred strains of mice. It is inherited as a single Mendelian factor with two alleles showing codominant expression. This structural gene, Bge, is closely linked (0/163 recombinants) with the Bgs site on chromosome 9 which regulates systemic levels of beta-galactosidase. The distribution of electrophoretic and activity phenotypes among inbred strains is not concordant, indicating that they result from separate mutations. Three aspects of beta-galactosidase realization, its structure, systemic regulation and developmental program, are now known to be organized in close proximity on chromosome 9. Considered in conjuction with evidence from other mammalian systems, this suggests that the mammalian genome, like Drosophila, is organized into large functional units in which relevant regulatory and developmental information is closely associated with individual structural genes.

Alleles↗
PubMed1977

Distribution of aryl hydrocarbon hydroxylase inducibility in cultured human lymphocytes.

We measured aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes. A striking seasonal variation in AHH activity was observed with induced AHH activity levels from January through May measuring approximately 20% of the values during the remainder of the year. AHH inducibility was determined by comparing lymphocytes from the same person cultured with and without the inducer 3-methylcholanthrene. If measurements are limited to the summer and fall seasons when AHH activity is high, AHH inducibility is reproducible for most persons with repeat determinations on the same person averaging 11% from the mean. The values of AHH inducibility in 53 persons ranged from 0.9 to 5.0, but the distribution of values did not fall into three distinct, nonoverlapping classes as reported by others. We were not able to determine the distribution of AHH inducibility in lung cancer patients since lymphocytes from less than half of the patients tested could be successfully cultured.

Aryl Hydrocarbon Hydroxylases↗
PubMed1976

Isolation, characterization, and radioimmunoassay of murine egasyn, a protein stabilizing glucuronidase membrane binding.

Glucuronidase present in lysosomes of mouse liver occurs as the free tetramer, whereas glucuronidase present in endoplasmic reticulum occurs in macromolecular complexes containing one to four molecules of the protein egasyn. Earlier genetic and biochemical studies suggest that these complexes, or M forms, function to stabilize the membrane binding of glucoronidase. The detergent Triton X-100 extracts glucuronidase-egasyn complexes intact and they dissociate in the presence of the detergent deoxycholate or upon heating. We have now purfied egasyn by releasing it from antiglucuronidase immunoprecipitates of M forms under relatively mild conditions, such as treatment with deoxycholate or heating at 50 degrees. Isolated egasyn is a glycoprotein of molecular weight about 64,000 and is not unusually hydrophobic in amino acid composition. Monospecific antibody to egasyn was raised. This antibody showed no cross-reactivity with purified beta-glucuronidase and antibody to glucuronidase failed to react with purified egasyn; however, both antibodies bound to egasyn-glucuronidase complexes. A procedure for the radioimmunoassay of egasyn was developed utilizing egasyn labeled with iodine 125. Most of the antigenic sites of egasyn in homogenates of normal liver are masked after extraction with Triton X-100 and only become immunoreactive after exposure to deoxycholate. After unmasking, mouse liver proved to contain about 56 mug of egasyn/g, nearly all of which is localized to the microsomal fraction. Of this total only about 10% was complexed with glucuronidase, suggesting theat the bulk of the egasyn present may be complexed with other proteins. Mice of the inbred strain YBR, which carry the EgO mutation resulting in the absence of microsomal glucuronidase, lacked immunoreactive egasyn, suggesting that the primary defect in this strain lies in the unavailabililty of agasyn to form complexes. There is now considerable evidence in support of the concept that the microsomal forms of glucuronidase exist in membranes complexed with egasyn and that formation of these complexes is required for maintenance of glucuronidase in membranes. Egasyn may represent one of a class of membrane anchor proteins that each stabilize the membrane binding of a charcteristic set of proteins.

Amino Acids↗
PubMed1976

Properties of mouse alpha-galactosidase.

alpha-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-alpha-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzmye was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a Km value of 1.4 mM, and energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human alpha-galactosidase A. The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the alpha-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.

Animals↗
PubMed1976

Genetic determination of the beta-galactosidase developmental program in mouse liver.

The developmental program for beta-galactosidase in C57BL/6J and related strains of mice differs from that seen in most other mouse strains. Mice of the C57BL/6 group show a rise in liver enzyme activity during development that is not seen in mice of other strains. The developmental pattern of beta-galactosidase activity in heart and brain of C57BL/6 mice is similar to that in other strains. Physical, kinetic, and immunological tests indicate that the developmental increase in C57BL/6 enzyme activity is an increase in the number of molecules of the same species of enzyme protein that is present in other strains. In genetic crosses, the presence of the developmental rise in liver enzyme activity segregates as if determined by a single genetic factor showing additive expression in heterozygotes. This factor is closely linked to the beta-galactosidase structural gene on chromosome 9. The existence of a genetic variant with these properties is evidence that programmatic information capable of regulating the temporal expression of a structural gene can be encoded in DNA.

Animals↗
PubMed1975

Purification and chemical properities of mouse liver lysosomal (L form) beta-glucuronidase.

The lysosomal form (L form) of beta-glucuronidase was purified 6,500-fold from the liver of C57BL/6J mice with high yield. Purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence or absence of sodium dodetcyl sulfate. The microsomal forms of beta-glucuronidase were spontaneously converted to the L form. The purified L form is a tetramer of molecular weight of 280,000 to 300,000, composedd of four identical subunits of 75,000 molecular weight. The enzyme contains a high content of arginine and glutamic acid and a very low content of sulfur-containing amino acids. Approximately 7% of the enzyme molecule is compose of carbohydrate. Sugars in the L form are glucosamine, mannose, galactose, and glucose. Sialic acid and fucose are absent in the enzyme.

Amino Acids↗
PubMed1975

Egasyn, a protein complexed with microsomal beta-glucuronidase.

Two polypeptide chains are present in murine beta-glucuronidase precipitated with a specific anti-beta-glucuronidase antibody F(ab)2 fragment. One is the catalytic subunit of beta-glucuronidase and the other has the properties predicted for the hypothetical beta-gluronidase membrane anchor protein. The new protein, named egasyn, is associated with microsomal, but no lysosomal beta-glucuronidase. It is released from the microsomal beta-glucuronidase complex by heat treatment. The YBR strain of mice carrying the Eg degrees mutation does not form an egasyn-beta-glucuronidase complex and is unable to retain beta-glucuronidase on microsomal membranes.

Animals↗
PubMed1975

The molecular genetics of mammalian glucuronidase.

The genetic factors known to be involved in the final realization of beta-glucuronidase activity in mice are considered from the standpoint of structural genes determining the catalytic activity of enzyme molecules as well as the recognition features of enzyme molecules that identify them for subsequent processing by the cell; processing genes determining the cellular apparatus involved with the conjugation, intracellular localization and eventual degradation of enzyme molecules; regulatory genes determining rates of enzyme synthesis, especially in response to physiological signals such as hormones; and temporal genes determining the developmental programs for expression of these classes during growth and differentiation. The properties of genetic variants of beta-glucuronidase falling into each of these classes are described. When those results are considered in concert with the properties of genetic variants known for other mammalian enzymes several generalizations emerge. Structural genes of enzymes are not usually linked to the processing genes determining the post-assembly events in the life of that enzyme. In contrast, all of the regulatory and temporal gene sites so far identified are in close proximity to the structural genes they modulate. Regulatory and temporal sites appear to act in a cis fashion to control the amount of enzyme synthesized from the adjacent structural allele on the same chromosome.

Animals↗