Huntington Porter

The particulate half-cystine-rich copper protein of newborn liver. Relationship to metallothionein and subcellular localization in non-mitochondrial particles possibly representing heavy lysosomes

Abstract The particulate half-cystine-rich copper protein of newborn liver can be partially purified by centrifugation of the heavy mitochondrial fraction through glycogen-sucrose or sucrose density gradients. The resulting sediments contained about 4% copper, about 20% half-cystine and a 2 to 3-fold increase in β-glucuronidase specific activity. The copper protein is not a true mitochondrial constituent and the data are consistent with its localization in a distinct population of heavy lysosomes. The amino acid composition of the polypeptide isolated from the crude insoluble copper protein is strikingly similar to that of metallothionein, suggesting that the neonatal protein represents a copper-rich form of metallothionein.

THE ISOLATION OF THE COPPER-CONTAINING PROTEIN CEREBROCUPREIN I FROM NORMAL HUMAN BRAIN*

PREVIOUS reports have described the separation from brain tissue under copper-free conditions of three different copper-containing fractions (PORTER and FOLCH, 1957a) and the isolation from one of them (bovine fraction I) of cerebrocuprein I, an essentially homogeneous brain protein containing 0.3 per cent copper (PORTER and FOLCH, 19576). Although the copper in the initial preparations of bovine arebrocuprein I appeared to react directly with sodium diethyldithiocarbamate, bovine cerebrocuprein I prepared by a milder method has subsequently been found to retain more than 90 per cent of its copper in competition with this reagent (PORTER and AWSWORTH, 1958). Since up to one-fourth or more of the pathological copper accumulating in the brains of patients with hepatolenticular degeneration appears to be bound in a form with properties similar to those of fraction I copper-protein(s) from normal human brains (PORTER, 1958), it seemed desirable to isolate cerebrocuprein I from normal human brain as a prerequisite to the determination, by immunological techniques, for example, of the amount of normal cerebrocuprein I present in the brains of patients with hepatolenticular degeneration. The present paper describes the isolation of cerebrocuprein I from normal human brain with further modifications in the isolation procedure.

The intracellular distribution of copper in immature liver.

Abstract The distribution of copper among subcellular fractions from immature and adult bovine liver has been determined. 1. 1. In immature liver with elevated total copper content, 46% of the tissue copper was found in the washed and combined mitochondrial fractions, 24% in the supernatant fraction, 21% in the washed nuclear fraction and 6% in the combined microsomal fraction. The finding of a large proportion of the copper of immature liver in the mitochondrial fractions suggests that the copper accumulating in immature liver may have specific enzymic function rather than merely furnishing “stored” copper to the immature animal. 2. 2. Copper per gram of fresh tissue in the washed and combined mitochondrial fractions from immature liver was more than five times that in the corresponding fractions from adult liver, more than ten times that in whole bovine heart, and more than forty times the copper previously found in mitochondrial fractions from adult bovine cerebral cortex. Since mitochondrial fractions from immature liver were low in cytochrome oxidase activity compared to whole bovine heart, it is concluded that immature liver contains a mitochondrial copper protein other than cytochrome oxidase.

NEONATAL HEPATIC MITOCHONDROCUPREIN. II. ISOLATION OF THE COPPER-CONTAINING SUBFRACTION FROM MITOCHONDRIA OF NEWBORN HUMAN LIVER.

The high concentration of copper occurring in newborn human liver, like that in newborn bovine liver, is localized predominantly in the mitochondrial fraction. The neonatal hepatic mitochondrocuprein subfraction, containing more than 2% copper, has been isolated from mitochondria of newborn human liver by the procedure previously developed for bovine liver. Yield of copper and concentration of copper in the subfraction from newborn human livers were more than 80 times those obtained in the corresponding subfraction from adult human livers. The human neonatal hepatic mitochondrocuprein subfraction was similar to that previously obtained from newborn bovine liver in its insolubility in the detergents used in the isolation procedure, in the quantitative release of its copper into soluble form by brief tryptic digestion, in the apparent stability of its copper-protein bond to a pH as acid as 1.5, and in the capacity of its copper to react directly with carbamate although at an extremely slow rate. The iron content of the human subfraction was relatively low and variable, suggesting that iron may not be an integral part of the human copper protein.

Human hepatocuprein: Isolation of a copper protein from the subcellular soluble fraction of adult human liver

Abstract A protein containing 0.3% copper was isolated from the subcellular soluble fraction of adult human liver. This human hepatocuprein is similar to human cerebrocuprein I and erythrocuprein in that it has a similar copper content, a sedimentation coefficient of the order of S 20 = 3S and a green color with a broad absorption maximum of low extinction at about 670 mμ. It is possible that the copper proteins of this type obtained from human liver, brain, and erythrocytes are identical. There is, however, some evidence suggesting that there may be some differences between the soluble copper proteins from the three different tissues.

Neonatal hepatic mitochondrocuprein I. Isolation of a protein fraction containing more than 4% copper from mitochondria of immature bovine liver

Abstract A protein fraction containing more than 4% copper has been isolated from mitochondria of immature bovine liver under copper-free conditions. This fraction retained 90% of its copper at a pH as acid as 1.5. At least 60% of its copper reacted directly with carbamate but at an extremenly slow rate. The mitochondrial copper was insoluble in a variety of solvents but could be dissolved by trypsin. The fraction also contained 2.4–3.0% iron but it is not certain that this iron is actually bound to the copper protein. Significant amounts of copper and iron were not found in the corresponding fraction prepared from adult liver.

Neonatal hepatic mitochondrocuprein: IV. Sulfitolysis of the cystine-rich crude copper protein and isolation of a peptide containing more than 35% half-cystine

Abstract Crude neonatal hepatic mitochondrocuprein, the detergent-insoluble copper protein accounting for the major proportion of the increased copper concentration occurring physiologically in the liver of newborn animals, has been prepared from mitochondria of newborn bovine liver by a modified procedure. These preparations contained 20–26% half-cystine as moles half-cystine per 100 moles total amino acids recovered, and more than 4% copper on a dry weight basis. Sulfitolysis of the insoluble crude copper protein solubilized 99% of the copper and about 75% of the protein. A polypeptide containing more than 35% half-cystine has been isolated from the solubilized material. The peptide gave a single peak on gel filtration on Sephadex G-100 in 6 M guanidine · HCl and appears to have a molecular weight between 5000 and 10 000. The uniquely high cystine content of the protein probably is the major factor in both the insolubility of the unsulfonated preparation and in its extraordinary capacity to bind copper in the intact newborn animal. It is suggested that the mitochondrial copper protein may function as a reservoir of copper for the formation of hepatic cytochrome oxidase during the neonatal period.

THE INTRACELLULAR DISTRIBUTION OF COPPER IN BRAIN

A PREVIOUS report (PORTER and FOLCH, 1957~) described the separation from brain tissue under copper-free conditions of three different copper-containing fractions: Fraction I, extracted from the fresh tissue with 0.1 M-acetate buffer pH 4.5 (or with water or with 0.1 hi-bicarbonate buffer pH 8-2); Fraction 11, obtained by subsequent extraction of the tissue residue with water at pH 3.5 and vanishing ionic strength; and the residual Fraction 111. Cerebrocuprein I, an essentially homogeneous brain protein containing 0.3% copper (PORTER and FOLCH, 19576), has been isolated both from bovine Fraction I and from normal human brain (PORTER and AINSWORTH, 1959) and shown to bind its copper in a form which does not react directly with sodium diethyldithiocarbamate (PORTER and AINSWORTH, 1958). Attempts to isolate the brain copper proteins in Fractions I1 and 111, however, have been unsuccessful. It seemed possible that the separation of particulate fractions by differential centrifugation might provide a more favourable starting point for the isolation of Fraction I1 and 111 copper proteins. The present paper describes the distribution of copper between cellular particulate fractions obtained from bovine cerebral cortex together with evidence that the major portion of the copper in the mitochondria1 fraction from brain is in the form previously designated as Fraction 111.