Ohkuma S, Chudzik J, Poole B
J Cell Biol. 1986 Mar;102(3):959-66. doi: 10.1083/jcb.102.3.959.
Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.
能提高培养的巨噬细胞溶酶体pH值的碱性物质和酸性离子载体（大隈修司和B. 普尔，1978年，《美国国家科学院院刊》，75:3327 - 3331；B. 普尔和大隈修司，1981年，《细胞生物学杂志》，90:665 - 669）抑制了细胞摄取的热变性乙酰化牛血清白蛋白（BSA）的消化。对于几种物质，pH值的变化足以解释蛋白水解的抑制作用。对于三丁胺、金刚烷胺和氯喹，必须假定存在其他可能对酶活性产生的影响。氟化钠（10 mM）对巨噬细胞分解BSA没有显著影响。10 mM氟化钠使内源性巨噬细胞蛋白质（无论是短寿命还是长寿命的）的分解受到约40%的抑制，而100 μM氯喹使其受到30%的抑制，对于长寿命蛋白质，抑制率有时更低。当细胞被提供BSA、细胞蛋白质混合物甚至惰性可内吞物质时，内源性长寿命蛋白质的分解以及氯喹对这一过程的抑制作用会选择性降低。细胞松弛素B或D对内吞作用的抑制并不影响氯喹敏感的内源性蛋白质分解，这表明通过该过程降解的蛋白质是真正内源性的，并非通过细胞自噬从外部摄取。另一方面，当巨噬细胞蛋白质在细胞外提供时，它们的分解对于短寿命和长寿命蛋白质以相同速率发生，并且强烈受到氯喹抑制而不受氟化钠抑制。从这些结果可以得出结论，内源性蛋白质（无论是短寿命还是长寿命的）的分解可能部分（约30%）发生在溶酶体中，部分通过一种或多种不受氯喹影响且可能易受氟化物抑制的非溶酶体机制发生。短寿命和长寿命蛋白质在到达溶酶体的方式或被这些细胞器处理的方式上必然存在差异；这种差异可以解释可内吞物质的供应对长寿命蛋白质溶酶体处理的选择性影响。
