Absorption of antisera for studies on specific enzyme turnover

J H Walker; S A Betts; R Manning; R J Mayer

doi:10.1042/bj1590355

Absorption of antisera for studies on specific enzyme turnover

Biochemical Journal ◽

10.1042/bj1590355 ◽

1976 ◽

Vol 159 (2) ◽

pp. 355-362 ◽

Cited By ~ 9

Author(s):

J H Walker ◽

S A Betts ◽

R Manning ◽

R J Mayer

Keyword(s):

Cytochrome Oxidase ◽

Early Stage ◽

General Procedure ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Phosphogluconate Dehydrogenase ◽

Tissue Extracts ◽

Enzyme Turnover ◽

Monospecific Antisera ◽

Mammary Gland Differentiation

Antisera were raised to acetyl-CoA carboxylase and 6-phosphogluconate dehydrogenase from mammary glands of lactating rabbits, and cytochrome oxidase from rat liver. The enzymes were all highly purified but gave rise to multispecific antisera when tested against tissue extracts. Absorption procedures were devised to free the antisera of contaminating antibodies. Antisera to acetyl-CoA carboxylase and cytochrome oxidase were absorbed with fractions discarded during enzyme purification. The antiserum to 6-phospho-gluconate dehydrogenase was absorbed with a tissue extract from an early stage in mammary-gland differentiation. Monospecific antisera are essential for enzyme turnover studies and therefore antisera should be extensively tested and absorbed before use. A general procedure for the absorption of antisera to purified enzymes has been devised on the basis of accepted principles of antisera absorption.

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Studies on acetyl-CoA carboxylase and fatty acid synthase from rat mammary gland and mammary tumours

Biochemical Journal ◽

10.1042/bj2080443 ◽

1982 ◽

Vol 208 (2) ◽

pp. 443-452 ◽

Cited By ~ 2

Author(s):

P M Ahmad ◽

D S Feltman ◽

F Ahmad

Keyword(s):

Mammary Gland ◽

Fatty Acid ◽

Fatty Acid Synthase ◽

Mammary Tissue ◽

Acetyl Coa Carboxylase ◽

Lipogenic Enzymes ◽

Acetyl Coa ◽

Rat Mammary Gland ◽

Mammary Gland Differentiation ◽

Mammary Adenocarcinomas

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase, were determined in two transplantable mammary adenocarcinomas (13762 and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammary tissue of control animals (non-tumour-carrying) of comparable physiological states. During mammary-gland differentiation of control or tumour-carrying animals, the activities of acetyl-CoA carboxylase and fatty acid synthase in the lactating gland increased by about 40-50-fold over the values found in non-pregnant animals. On the other hand, in tumours carried by lactating dams there were only modest increases (1.5-2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with the neoplasms carried by non-pregnant animals. On the basis of the Km values for different substrates and immunodiffusion and immunotitration data, the fatty acid synthase of neoplastic tissues appeared to be indistinguishable from the control mammary-gland enzyme. However, a comparison of the immunotitration and immunodiffusion experiments indicated that the mammary-gland acetyl-CoA carboxylase might differ from the enzyme present in mammary neoplasms.

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Variations in the activity of several enzymes in the mammary glands of non-pregnant, pregnant and lactating rabbits

Biochemical Journal ◽

10.1042/bj1160657 ◽

1970 ◽

Vol 116 (4) ◽

pp. 657-661 ◽

Cited By ~ 8

Author(s):

P. E. Hartmann ◽

E. A. Jones

Keyword(s):

Mammary Gland ◽

Small Mammals ◽

Enzyme Activities ◽

Mammary Glands ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Phosphogluconate Dehydrogenase ◽

Citrate Lyase ◽

Lactation Cycle

1. The enzymes phosphofructokinase (EC 2.7.1.11), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), ATP–citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) were assayed in rabbit mammary glands at various stages of the pregnancy–lactation cycle. 2. The activities of all enzymes were low during pregnancy and, with the exception of phosphofructokinase, in non-pregnant animals. Two- to ten-fold increases in enzyme activities occurred over the first 20 days of lactation. Although milk yield was considerably decreased, the enzyme activities remained elevated in late lactation (45 days after parturition). 3. These findings are discussed in relation to mammary-gland metabolism and compared with similar observations previously made on ruminants and other small mammals.

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Evidence that insulin activates fat-cell acetyl-CoA carboxylase by increased phosphorylation at a specific site

Biochemical Journal ◽

10.1042/bj2020077 ◽

1982 ◽

Vol 202 (1) ◽

pp. 77-86 ◽

Cited By ~ 83

Author(s):

R W Brownsey ◽

R M Denton

Keyword(s):

Covalent Modification ◽

Performic Acid ◽

Specific Site ◽

Kinetic Properties ◽

Fat Cells ◽

Acetyl Coa Carboxylase ◽

Fat Cell ◽

Acetyl Coa ◽

Fresh Tissue ◽

Tissue Extracts

1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].

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